Peptides for treatment and prevention of nonalcoholic fatty liver disease and fibrosis

ABSTRACT

The present invention relates to peptides for the treatment or prevention of nonalcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), hepatic steatosis (fatty liver), liver inflammation, cirrhosis, hepatocellular carcinoma or fibrosis, especially liver fibrosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/772,178, filed Jun. 12, 2020, now U.S. Pat. No. 11,530,241, which isthe U.S. national stage application of International Patent ApplicationNo. PCT/EP2018/085071, filed Dec. 14, 2018.

The Sequence Listing for this application is labeled“Seq-List-replace.xml” which was created on May 26, 2023 and is 86,519bytes. The entire content of the sequence listing is incorporated hereinby reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of the medicine. Moreparticularly, it relates to treatment of liver diseases, in particularhepatic steatosis, especially non-alcoholic steatotic hepatitis and totreatment of fibrosis.

BACKGROUND OF THE INVENTION

NAFLD (nonalcoholic fatty liver disease), defined by the presence ofhepatic accumulation of triglycerides in the hepatocytes in absence ofany other etiology of liver disease, is the most common cause of chronicliver disease in the Western world. Its clinical-histologic phenotypeextends from nonalcoholic fatty liver (NAFL) to nonalcoholicsteatohepatitis (NASH), characterized by liver inflammation andprogressive fibrosis, leading to cirrhosis and end stage liver diseaseas well as hepatocellular carcinoma.

Whereas the estimated prevalence of NAFLD ranges from 6 to 33% in thegeneral population, the prevalence of NASH only ranges from 3 to 5%, butNASH-related cirrhosis has become the second leading indication forliver transplantation in the United States. Hospitalizations for NAFLDhave increased by 97% since the year 2000.

There are no drugs currently approved to prevent or treat NAFLD or NASH.

Therefore, there is a need for new treatments for preventing or treatingnonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis(NASH), and hepatic steatosis (fatty liver).

Fibrosis is a pathological condition where fibrous connective tissueinvades any organ, usually as a consequence of inflammation or otherinjury. Several compounds are known to treat fibrosis but do soinadequately. Thus, attempts to develop a clinically effective fibrosishave been unsuccessful, and there is still a need to find treatments forfibrosis.

SUMMARY OF THE INVENTION

Surprisingly, the inventors provide peptides from the kinase domain ofthe PKCα and derivatives thereof which specifically decrease theexpression of Solute Carrier Family 27 Member 2 (SLC27A2) commonly knownas FATP2 (Fatty acid transport protein 2) in adipose tissue. Thepeptides are capable, after 3 months of a single injection, ofdecreasing the phenomenon of steatosis on the liver, in particularcapable of decreasing the size of the lipid droplets in the liver, thelevel of two biomarkers of liver damage (i.e., AST and ALT) and theratio of liver weight to body weight. In addition, the peptides arecapable of decreasing fibrosis, as shown by their capacity todown-regulate de novo lipogenesis pathway (e.g., decrease of ACC(Acetyl-CoA carboxylase) and to decrease LOXL2 hepatic protein contentand circulating levels, thereby stopping fibrosis progression. In vivo,a significant decrease of liver triglycerides content and fibrosis areahave been observed with a treatment with the peptides, demonstrating ananti-steatotic and anti-fibrosis effect.

Accordingly, the present invention relates to a peptide, wherein

-   -   the peptide is capable of decreasing the FATP2 expression in        adipose tissue, in particular in a mammal;    -   the peptide does not simultaneously comprise one methionine, one        proline and one arginine;    -   the peptide adopts a secondary structure which is a helix,        preferably an alpha helix; and    -   the peptide comprises, consists essentially in or consists in a        sequence from a segment of at least 5, 6, 7, 8, 9, 10, 11, 12,        13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 25 consecutive        residues of the kinase domain of a PKC (Protein Kinase C) or a        segment from 5 to 40 consecutive residues of the kinase domain        of a PKC (Protein Kinase C);    -   the peptide has a length from 5 to 80 amino acids or from 5 to        60 amino acids or from 5 to 40 amino acids, and    -   the peptide sequence may comprise 1, 2, 3, 4, or 5        modification(s) of an amino acid selected from substitution(s),        deletion(s), addition(s), and a mixture thereof within said        sequence of a segment of the kinase domain of the PKC.

Preferably, the peptide is modified by a chemical cross-linking processsuch as stapling.

Preferably, the peptide has a length of at least 5 amino acids and lessthan 40 amino acids, preferably a length of at least 5 amino acids andless than 30 amino acids, more preferably of at least 5 amino acids andless than 25 amino acids.

Preferably, the peptide is capable of decreasing or preventing theinteraction between ALMS1 and αPKC.

Optionally, the peptide sequence comprises, consists essentially in orconsists in at least one of the following sequences: VECTMVEKRVLA (SEQID NO: 3) with optionally 1, 2, 3, 4, or 5 modification(s) of an aminoacid selected from substitution(s), deletion(s), addition(s), and amixture thereof, more preferably, 1, 2, or 3 modification(s) of an aminoacid selected from substitution(s) deletion(s), addition(s), and amixture thereof; VECTXVEKRVLA (SEQ ID NO: 9) with optionally 1, 2, 3, 4,or 5 modification(s) of an amino acid selected from substitution(s),deletion(s), addition(s), and a mixture thereof, more preferably, 1, 2,or 3 modification(s) of an amino acid selected from substitution(s)deletion(s), addition(s), and a mixture thereof; VECTMVEKXVLA (SEQ IDNO: 10) with optionally 1, 2, 3, 4, or 5 modification(s) of an aminoacid selected from substitution(s), deletion(s), addition(s), and amixture thereof, more preferably, 1, 2, or 3 modification(s) of an aminoacid selected from substitution(s) deletion(s), addition(s), and amixture thereof; VECTXVEKXVLA (SEQ ID NO: 11) with optionally 1, 2, 3,4, or 5 modification(s) of an amino acid selected from substitution(s),deletion(s), addition(s), and a mixture thereof, more preferably, 1, 2,or 3 modification(s) of an amino acid selected from substitution(s)deletion(s), addition(s), and a mixture thereof; LMYHIQQV (SEQ ID NO: 4)with optionally 1, 2, or 3 modification(s) of an amino acid selectedfrom substitution(s) deletion(s), addition(s), and a mixture thereof;LXYHIQQV (SEQ ID NO: 12) with optionally 1, 2, or 3 modification(s) ofan amino acid selected from substitution(s) deletion(s), addition(s),and a mixture thereof; LDN; SVDWWAYGVLLYEMLA (SEQ ID NO: 6) withoptionally 1, 2, 3, 4, or 5 modification(s) of an amino acid selectedfrom substitution(s), deletion(s), addition(s), and a mixture thereof,more preferably, 1, 2, or 3 modification(s) of an amino acid selectedfrom substitution(s) deletion(s), addition(s), and a mixture thereof;SVDWWAYGVLLYEXLA (SEQ ID NO: 13) with optionally 1, 2, 3, 4, or 5modification(s) of an amino acid selected from substitution(s),deletion(s), addition(s), and a mixture thereof, more preferably, 1, 2,or 3 modification(s) of an amino acid selected from substitution(s)deletion(s), addition(s), and a mixture thereof; EDEDELFQSIME (SEQ IDNO: 7) with optionally 1, 2, 3, 4, or 5 modification(s) of an amino acidselected from substitution(s), deletion(s), addition(s), and a mixturethereof, more preferably, 1, 2, or 3 modification(s) of an amino acidselected from substitution(s) deletion(s), addition(s), and a mixturethereof; EDEDELFQSIXE (SEQ ID NO: 14) with optionally 1, 2, 3, 4, or 5modification(s) of an amino acid selected from substitution(s),deletion(s), addition(s), and a mixture thereof, more preferably, 1, 2,or 3 modification(s) of an amino acid selected from substitution(s)deletion(s), addition(s), and a mixture thereof; GERDVRE (SEQ ID NO: 8)with optionally 1, 2, or 3 modification(s) of an amino acid selectedfrom substitution(s) deletion(s), addition(s), and a mixture thereof;GEXDVRE (SEQ ID NO: 15) with optionally 1, 2, or 3 modification(s) of anamino acid selected from substitution(s) deletion(s), addition(s), and amixture thereof; GERDVXE (SEQ ID NO: 16) with optionally 1, 2, or 3modification(s) of an amino acid selected from substitution(s)deletion(s), addition(s), and a mixture thereof; GEXDVXE (SEQ ID NO: 17)with optionally 1, 2, or 3 modification(s) of an amino acid selectedfrom substitution(s) deletion(s), addition(s), and a mixture thereof;LDN; AFF; PDY; XDY; PEII (SEQ ID NO: 5); XEII (SEQ ID NO: 18); PAK; XAK;wherein X is any amino acid except M, P and R.

Optionally, the peptide sequence comprises, consists essentially in orconsists in at least one of the following sequences: VECTMVEKRVLA (SEQID NO: 3) with optionally 1, 2, or 3 modification(s) of an amino acidselected from substitution(s) deletion(s), addition(s), and a mixturethereof; VECTXVEKRVLA (SEQ ID NO: 9) with optionally 1, 2, or 3modification(s) of an amino acid selected from substitution(s)deletion(s), addition(s), and a mixture thereof; VECTMVEKXVLA (SEQ IDNO: 10) with optionally 1, 2, or 3 modification(s) of an amino acidselected from substitution(s) deletion(s), addition(s), and a mixturethereof; VECTXVEKXVLA (SEQ ID NO: 11) with optionally 1, 2, or 3modification(s) of an amino acid selected from substitution(s)deletion(s), addition(s), and a mixture thereof; LMYHIQQV (SEQ ID NO: 4)with optionally 1, 2, or 3 modification(s) of an amino acid selectedfrom substitution(s) deletion(s), addition(s), and a mixture thereof;LXYHIQQV (SEQ ID NO: 12) with optionally 1, 2, or 3 modification(s) ofan amino acid selected from substitution(s) deletion(s), addition(s),and a mixture thereof; SVDWWAYGVLLYEMLA (SEQ ID NO: 6) with optionally1, 2, or 3 modification(s) of an amino acid selected fromsubstitution(s) deletion(s), addition(s), and a mixture thereof;SVDWWAYGVLLYEXLA (SEQ ID NO: 13) with optionally 1, 2, or 3modification(s) of an amino acid selected from substitution(s)deletion(s), addition(s), and a mixture thereof; EDEDELFQSIME (SEQ IDNO: 7) with optionally 1, 2, or 3 modification(s) of an amino acidselected from substitution(s) deletion(s), addition(s), and a mixturethereof; EDEDELFQSIXE (SEQ ID NO: 14) with optionally 1, 2, or 3modification(s) of an amino acid selected from substitution(s)deletion(s), addition(s), and a mixture thereof; GERDVRE (SEQ ID NO: 8)with optionally 1, 2, or 3 modification(s) of an amino acid selectedfrom substitution(s) deletion(s), addition(s), and a mixture thereof;GEXDVRE (SEQ ID NO: 15) with optionally 1, 2, or 3 modification(s) of anamino acid selected from substitution(s) deletion(s), addition(s), and amixture thereof; GERDVXE (SEQ ID NO: 16) with optionally 1, 2, or 3modification(s) of an amino acid selected from substitution(s)deletion(s), addition(s), and a mixture thereof; GEXDVXE (SEQ ID NO: 17)with optionally 1, 2, or 3 modification(s) of an amino acid selectedfrom substitution(s) deletion(s), addition(s), and a mixture thereof;wherein X is any amino acid except M, P and R.

Optionally, the peptide sequence comprises, consists essentially in orconsists in at least one of the following sequences:

-   -   a) VECTXVEKXVLALLDKXXFLTQLHS (SEQ ID NO: 20) wherein X is any        amino acid except M, P and R, preferably, an amino acid        favorable to an α-helix secondary structure, more preferably        selected from the group consisting of A, D, N, C, G, Q, E, H, L,        K, F, S, W and Y, still more preferably A, D, N, G, Q, E, H, L,        K, F, S, W and Y, with optionally 1, 2, 3, 4, or 5        modification(s) of an amino acid selected from substitution(s),        deletion(s), addition(s), and a mixture thereof, more        preferably, 1, 2, or 3 modification(s) of an amino acid selected        from substitution(s) deletion(s), addition(s), and a mixture        thereof;    -   b) VECTMVEKRVLALLDKXXFLTQLHS (SEQ ID NO: 21) wherein X is any        amino acid except M, P and R, preferably, an amino acid        favorable to an α-helix secondary structure, more preferably        selected from the group consisting of A, D, N, C, G, Q, E, H, L,        K, F, S, W and Y, still more preferably A, D, N, G, Q, E, H, L,        K, F, S, W and Y, with optionally 1, 2, 3, 4, or 5        modification(s) of an amino acid selected from substitution(s),        deletion(s), addition(s), and a mixture thereof, more        preferably, 1, 2, or 3 modification(s) of an amino acid selected        from substitution(s) deletion(s), addition(s), and a mixture        thereof;    -   c) VECTXVEKRVLALLDKPPFLTQLHS (SEQ ID NO: 22) wherein X is any        amino acid except M, P and R, preferably, an amino acid        favorable to an α-helix secondary structure, more preferably        selected from the group consisting of A, D, N, C, G, Q, E, H, L,        K, F, S, W and Y, still more preferably A, D, N, G, Q, E, H, L,        K, F, S, W and Y, with optionally 1, 2, 3, 4, or 5        modification(s) of an amino acid selected from substitution(s),        deletion(s), addition(s), and a mixture thereof, more        preferably, 1, 2, or 3 modification(s) of an amino acid selected        from substitution(s) deletion(s), addition(s), and a mixture        thereof;    -   d) VECTMVEKXVLALLDKPPFLTQLHS (SEQ ID NO: 23) wherein X is any        amino acid except M, P and R, preferably, an amino acid        favorable to an α-helix secondary structure, more preferably        selected from the group consisting of A, D, N, C, G, Q, E, H, L,        K, F, S, W and Y, still more preferably A, D, N, G, Q, E, H, L,        K, F, S, W and Y, with optionally 1, 2, 3, 4, or 5        modification(s) of an amino acid selected from substitution(s),        deletion(s), addition(s), and a mixture thereof, more        preferably, 1, 2, or 3 modification(s) of an amino acid selected        from substitution(s) deletion(s), addition(s), and a mixture        thereof; and the sequence of any segment of at least 5, 6, 7, 8,        9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 25        consecutive residues of any sequence a) to d).

Optionally, the peptide sequence comprises, consists essentially in orconsists in at least one of the following sequences:

(SEQ ID NO: 24) VECTM X EKRVLAX (SEQ ID NO: 25) VECTX X EKRVLA X(SEQ ID NO: 26) VECTM X EKXVLA X (SEQ ID NO: 27) VECTX X EKXVLA X(SEQ ID NO: 28) VECTX X EKXVLA X LDKXXFLTQLHS (SEQ ID NO: 29) VECTM XEKRVLA X LDKXXFLTQLHS (SEQ ID NO: 30) VECTX X EKRVLA X LDKPPFLTQLHS(SEQ ID NO: 31) VECTM X EKXVLA X LDKPPFLTQLH

-   -   wherein the residues which are bold and underlined X carry the        stapling and is any amino acid derivative suitable for stapling;        and    -   wherein X is any amino acid except M, P and R,    -   with the sequence having optionally 1, 2, 3, 4, or 5        modification(s) of an amino acid selected from substitution(s),        deletion(s), addition(s), and a mixture thereof, more        preferably, 1, 2, or 3 modification(s) of an amino acid selected        from substitution(s) deletion(s), addition(s), and a mixture        thereof.

Preferably, said PKC is selected from the group consisting of analpha-PKC (αPKC), a beta-PKC (βPKC) including βI and βII PKC, delta-PKC,theta-PKC, eta-PKC and epsilon-PKC. More preferably, said PKC is an αPKCof SEQ ID NO: 1.

In a particular embodiment, the peptide sequence comprises, consistsessentially in or consists in

(SEQ ID NO: 32) VECTT R EKEVLA S LDKAAFLTQLHS

-   -   wherein R and S carry the stapling, being preferably        2-(7-octenyl)arginine and 2-(4-pentenyl)serine, respectively;    -   with the sequence having optionally 1, 2, 3, 4, or 5        modification(s) of an amino acid selected from substitution(s),        deletion(s), addition(s), and a mixture thereof, more        preferably, 1, 2, or 3 modification(s) of an amino acid selected        from substitution(s) deletion(s), addition(s), and a mixture        thereof.

The present invention also relates to a pharmaceutical compositioncomprising a peptide according to the present disclosure. It alsorelates to a peptide according to the present disclosure for use as adrug. It further relates to the use of a peptide according to thepresent disclosure for the manufacture of a drug.

It further relates to a peptide according to the present disclosure or apharmaceutical composition comprising it for use in the treatment orprevention of a disease selected from the group consisting ofnonalcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver(NAFL), non-alcoholic steatohepatitis (NASH), hepatic steatosis (fattyliver), liver inflammation, cirrhosis, hepatocellular carcinoma andfibrosis.

Optionally, the disease is selected from the group consisting ofnonalcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver(NAFL), non-alcoholic steatohepatitis (NASH), and hepatic steatosis(fatty liver). Preferably, the disease is hepatic steatosis (fattyliver) or non-alcoholic steatohepatitis (NASH). More preferably, thedisease is non-alcoholic steatohepatitis (NASH).

Optionally, the fibrosis is a liver fibrosis including cirrhosis, arenal fibrosis, a cardiac fibrosis including an atrial fibrosis, anendomyocardial fibrosis and old myocardial infarction, a pulmonaryfibrosis including cystic fibrosis and radio-induced lung fibrosis, avascular fibrosis such as an arterial fibrosis, a brain fibrosis, amyelofibrosis, an arthrofibrosis, an intestinal fibrosis, a peritonealfibrosis, a retroperitoneal fibrosis or a skin fibrosis. Preferably, thefibrosis is a liver fibrosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C: Effect of adipose tissue targeted PATAD treatment onexpression levels of key fatty acids transporters and receptors.

n=4 animals per group, GAPDH was used as reference gene. There are 6isoforms of FATP (FATP1-6 also known as SLC27A1-SLC27A6).

Fatp1 (Fatty acid transport protein 1), Fatp2 (Fatty acid transportprotein 2), Fatp3 (Fatty acid transport protein 3), Fatp4 (Fatty acidtransport protein 4), Fatp5 (Fatty acid transport protein 5), Fatp6(Fatty acid transport protein 6), FFAR1 (Free Fatty Acid Receptor 1),FFAR2 (Free Fatty Acid Receptor 2), FFAR3 (Free Fatty Acid Receptor 3),FFAR4 (Free Fatty Acid Receptor 4).

“Scramble less” refers to the combination of the two peptides with thesame amino acid residues as in the stapled forms but randomly rearrangedwith a maintained alpha helix structure: Scrambled peptide sequence Aand Scrambled peptide sequence B. “Staple” refers to a combination oftwo stapled peptides: Stapled peptide sequence A and Stapled peptidesequence B.

FIG. 1A depicts the expression levels of the FATPs and FFARs isoforms inthe PATAD injected adipose tissue where FATP2 expression level issignificantly reduced.

FIGS. 1B and 1C depict the expression levels of the FATPs and FFARsisoforms in the liver and muscle following subcutaneous PATAD injection.

FIGS. 2A-2B: ADPIF peptide is more active than PATAD peptide in reducingFATP2 expression level in the adipose tissue and in increasing GLP-1circulating levels.

FIG. 2A: 4 month-old male mice fed with a high fat/high glucose dietwere used in this set of experiment. Normalized expression levels ofFATP2 in the adipose tissue from the different mice 11 days post theindicated condition on the X-axis. N=4 animals per group, GAPDH was usedas reference gene.

FIG. 2B: Circulating concentration of GLP-1 in the same mice 11 dayspost injection showing that PATAD and ADPIF were able to restore GLP1circulating levels back to lean controls where scrambled injected micepresented decreased GLP-1 concentrations.

FIGS. 3A-3D: Effect on liver and circulating aspartate transaminase(AST) and alanine transaminase (ALT) after 3 months of adipose tissuetargeted PATAD and ADPIF treatment.

FIG. 3A: Fluorescent staining of cryosections from fixated liver fromcontrol (left) and PATAD (right) treated mice 3 months after one uniquePATAD injection.

FIG. 3B: Ratio of liver versus body weight times age in days was plottedfor n=1 animal per group for PATAD peptide.

FIGS. 3C-3D: Mean values of both AST (FIG. 3C) and ALT (FIG. 3D)measured by ELISA approach in plasma of mice with the indicatedtreatment. N=4 samples per group.

PATAD and ADPIF are both effective in reducing circulating levels of ASTand ALT. ADPIF is more active in reducing circulating levels of ALT,which translates improvement of liver cell injury.

FIGS. 4A-4B: ADPIF peptide is more active than PATAD peptide inpreventing hyperglycemia.

FIG. 4A: Time point series in days at 30 minutes glucose bolus infasting male diet induced obese (DIO) (CTL HFD) and controls (CTL Chowdiet) after a single injection of the PATAD (PATAD 417) or ADPIF (CPCpeptide A-MRP) treatment at day 0. The glucose bolus is injectedsubcutaneously which by-passes the liver and goes directly in the bloodstream.

FIG. 4B: Effect on glucose tolerance in fasting male diet induced obese(DIO) (CTL HFD) and controls (CTL Chow diet) by measure of the areaunder the curve before (D-5) at the moment of treatment and after (D+6)treatment with PATAD (PATAD 417) and ADPIF (PATAD 417-MRP) peptides.

FIGS. 5A-5B: ADPIF is more active than PATAD in decreasing hepaticprotein content of lysyl oxidase like 2 protein (LOXL2) and fatty acidbinding protein 4 (FABP4) in DIO male mice.

FIG. 5A: LOXL2 is a known key actor for fibrosis progression. ELISAresults measuring LOXL2 on liver extracts 3 months post vehicle, PATADor ADPIF injection in the subcutaneous adipose tissue. A significantdecrease in hepatic protein content is observed after either PATAD orADPIF treatment with ADPIF being the most effective peptide. N=4 miceper group.

FIG. 5B: FABP4 is a key actor of lipid-mediated processes in the celland is elevated in the liver associated with NAFLD. We measured theFABP4 protein content 3 months after either scramble or PATAD or ADPIFsingle injection. FABP4 is significantly reduced following either PATADor ADPIF injection, with ADPIF being more active than PATAD.

FIG. 6 . Effect of ADPIF on ceramides in the liver content and profile.Ceramides are a group of biological active lipids known to be involvedin NAFLD. We measured and determined the effect of ADPIF injections onthe ceramides profile following 3 injections of ADPIF at a frequency ofone injection per week in the subcutaneous adipose tissue. ADPIFglobally induces a decrease in the hepatic ceramides content withvariations between the different ceramides.

FIG. 7 . Effect on the liver triglycerides content after 3 months ofadipose tissue targeted ADPIF treatment.

Fluorescent staining of cryosections from fixated liver from control(left) and ADPIF (right) treated mice 3 months after one unique ADPIFinjection showing a decrease in the diameter of the lipid dropletsindicating an overall decrease in total triglycerides in the liver. Micewere DIO male mice age 7 months at the end of the experiment.

FIG. 8 . ADPIF peptide treatment downregulates de novo lipogenesispathway genes after 11 days in DIO male mice.

11-day post ADPIF peptide injection in the adipose tissue, hepaticexpression levels of fatty acid synthase (Fasn), Acetyl-CoA carboxylase(Acc) and Sterol regulatory element binding transcription factor 1(Srebf1) were significantly decreased compared to control indicatingthat the de novo lipogenesis in the liver of ADPIF-treated mice was shutdown.

FIGS. 9A-9C: ADPIF treatment reduces fibrotic lesions in the liver.

FIG. 9A: Immunostaining pictures of cryosections from liver of DIO-NASHuntreated and DIO-NASH treated with ADPIF, 3 months after ADPIFinjection in the subcutaneous adipose tissue. Collagen IV and LOXL2 ishighly expressed in the DIO-NASH untreated and reduced in the DIO-NASHADPIF treated.

FIG. 9B: As LOXL2 is also secreted, we measured the effect of ADPIF onthe circulation levels of LOXL2 and found that ADPIF treatment (3 monthspost a unique injection) reduced the LOXL2 levels by half.

FIG. 9C: Liver sections were analyzed by transmitted electron microscopyto identify the fibrotic depots in the DIO-NASH untreated liver. No suchfibrotic depots were found in the DIO-NASH treated liver 3 months afterADPIF injection in the subcutaneous adipose tissue.

FIGS. 10A-10D: ADPIF treatment impact on lysophosphatidylcholines (LPC)lipids.

Lysophosphatidylcholines (LPCs) are substrates for a pro-fibroticenzymes, Autotaxin which is a key enzyme in the generation oflysophosphatidic acid. The latter is a bioactive lipid known to beplaying a role in fibrotic progression that affect the liver but alsothe kidney and other soft tissues. We therefore measured the level ofthe different (LPCs) in pancreas (FIG. 10A), adipose tissue (FIG. 10B),liver (FIG. 10C) and plasma (FIG. 10D).

ADPIF treatment (given at a frequency of one subcutaneous injection of25 μg per mouse for a period of 3 weeks and then the mice wereeuthanized after 1 week after the last injection), induces a selectivedecrease of certain LPCs in the different tissues analyzed. LPC 18:2 isthe lipid which is the mostly reduced following ADPIF treatment both inthe pancreas, adipose tissue and liver.

FIG. 11 : ADPIF peptide protects the kidney from fibrosis.

Cryosections from kidneys of DIO-NASH treated with vehicle and DIO-NASHADPIF treated were immunostained for Collagen IV, ZO-1 and the nuclei.More depots of Collagen IV are observed in the DIO-NASH vehicle than inthe ADPIF treated.

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, the inventors provide peptides from the kinase domain ofthe PKCα and derivatives thereof which specifically decrease theexpression of FATP2 (Fatty acid transport protein 2) in adipose tissue(FIG. 1A). The peptides are capable, after 3 months of a singleinjection, of decreasing the phenomenon of steatosis on the liver, inparticular capable of decreasing the size of the lipid droplets in theliver, the level of two biomarkers of liver damage (i.e., AST and ALT)and the ratio of liver weight to body weight (FIG. 3 ). In addition, thepeptides are capable of decreasing fibrosis, as shown by their capacityto down-regulate de novo lipogenesis pathway (e.g., decrease of ACC(Acetyl-CoA carboxylase) (FIG. 8 ) and to decrease the protein level ofLOXL2 in the liver and in circulation (FIGS. 5 and 9A), thereby stoppingfibrosis progression. The peptides are also capable of decreasingcollagen deposit in the kidney. In vivo, a significant decrease offibrosis area has been observed with a treatment with the peptides,demonstrating an anti-fibrosis effect.

Accordingly, the invention relates to

-   -   a peptide as defined herein;    -   a pharmaceutical composition comprising a peptide as defined        herein;    -   a peptide as defined herein for use as a drug or the use of a        peptide as defined herein for the manufacture of a drug;    -   a peptide or a pharmaceutical composition comprising the peptide        for use in the treatment or prevention of a disease selected        from the group consisting of nonalcoholic fatty liver disease        (NAFLD), non-alcoholic fatty liver (NAFL), non-alcoholic        steatohepatitis (NASH), hepatic steatosis (fatty liver), liver        inflammation, cirrhosis, hepatocellular carcinoma and fibrosis,        especially a fibrosis selected from the group consisting of a        liver fibrosis including cirrhosis, a renal fibrosis, a cardiac        fibrosis including an atrial fibrosis, an endomyocardial        fibrosis and old myocardial infarction, a pulmonary fibrosis        including cystic fibrosis and radio-induced lung fibrosis, a        vascular fibrosis such as an arterial fibrosis, a brain        fibrosis, a myelofibrosis, an arthrofibrosis, an intestinal        fibrosis, a peritoneal fibrosis, a retroperitoneal fibrosis and        a skin fibrosis;    -   the use of a peptide for the manufacture of a medicine for the        treatment or prevention of a disease selected from the group        consisting of nonalcoholic fatty liver disease (NAFLD),        non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis        (NASH), hepatic steatosis (fatty liver), liver inflammation,        cirrhosis, hepatocellular carcinoma and fibrosis, especially a        fibrosis selected from the group consisting of a liver fibrosis        including cirrhosis, a renal fibrosis, a cardiac fibrosis        including an atrial fibrosis, an endomyocardial fibrosis and old        myocardial infarction, a pulmonary fibrosis including cystic        fibrosis and radio-induced lung fibrosis, a vascular fibrosis        such as an arterial fibrosis, a brain fibrosis, a myelofibrosis,        an arthrofibrosis, an intestinal fibrosis, a peritoneal        fibrosis, a retroperitoneal fibrosis and a skin fibrosis;    -   a method for the treatment or prevention of a disease in a        subject, comprising administering a therapeutically effective        amount of a peptide, wherein the disease is selected from the        group consisting of nonalcoholic fatty liver disease (NAFLD),        non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis        (NASH), hepatic steatosis (fatty liver), liver inflammation,        cirrhosis, hepatocellular carcinoma and fibrosis, especially a        fibrosis selected from the group consisting of a liver fibrosis        including cirrhosis, a renal fibrosis, a cardiac fibrosis        including an atrial fibrosis, an endomyocardial fibrosis and old        myocardial infarction, a pulmonary fibrosis including cystic        fibrosis and radio-induced lung fibrosis, a vascular fibrosis        such as an arterial fibrosis, a brain fibrosis, a myelofibrosis,        an arthrofibrosis, an intestinal fibrosis, a peritoneal        fibrosis, a retroperitoneal fibrosis and a skin fibrosis.

Definitions

ALMS1, Alström syndrome protein 1, is a protein encoded by the ALMS1gene. Mutations in the ALMS1 gene have been found to be causative forAlström syndrome. It is described in several databases, namely UniProtID No Q8TCU4; Gene ID No 7840, HGNG ID No 428. Reference sequences aredisclosed in Genbank under NM_015120.4 for mRNA and NP 055935.4 forprotein.

The terms “Protein kinase C” and “PKC” (EC 2.7.11.13) are equivalent andrefers to a family of protein kinase enzymes that are involved incontrolling the function of other proteins through the phosphorylationof hydroxyl groups of serine and threonine amino acid residues on theseproteins. PKC are typically activated by signals such as increases inthe concentration of diacylglycerol (DAG) or calcium ions (Ca2+). PKCplay important roles in several signal transduction cascades.

The PKC family comprises at least fifteen isozymes in humans, dividedinto three main subfamilies, conventional (or classical) PKCs, novelPKCs, and atypical PKCs.

Conventional (c)PKCs comprises the isoforms α, βI, βII, and γ. ThesePKCs require Ca²⁺, DAG, and a phospholipid such as phosphatidylserinefor activation.

Novel (n)PKCs include the δ, ε, η, and θ isoforms. These PKCs requireDAG, but do not require Ca2⁺ for activation.

Atypical (a)PKCs include the ζ, ι, and λ isoforms. These PKCs requireneither Ca2+ nor diacylglycerol for activation.

Protein kinase C alpha type, also called αPKC, PKC-A or PKC-alpha,belongs to a family of serine- and threonine-specific protein kinasesthat can be activated by calcium and the second messengerdiacylglycerol. It is described in several databases, namely UniProt IDNo P17252, Gene ID No 9393, HGNG ID No 5578. Reference sequences aredisclosed in Genbank under NM_02737.2 for mRNA and NP_002728.1 forprotein. The protein sequence of human αPKC is disclosed in SEQ ID NO:1.

The kinase domain of the αPKC is from position 339 to position 595 asdisclosed in SEQ ID NO: 1 and is shown in SEQ ID NO: 2.

“consists of,” “consists essentially of” or “substantially comprises”:The description herein of any aspect or embodiment of the inventionusing terms such as reference to an element or elements is intended toprovide support for a similar aspect or embodiment of the invention that“consists of,” “consists essentially of” or “substantially comprises”that particular element or elements, unless otherwise stated or clearlycontradicted by context. For instance, a peptide or protein describedherein as comprising a particular sequence should be understood as alsodescribing a peptide or protein consisting of that sequence, unlessotherwise stated or clearly contradicted by context. By “consistsessentially of” is intended that the peptide or protein consists of thatsequence, but it may also include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10substitutions, additions, deletions or a mixture thereof, preferably 1,2, 3, 4, or 5 substitutions, additions, deletions or a mixture thereof.In particular, by “essentially consist in”, it may be intended that thepeptide may include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional aminoacids at the N and/or C-terminal end, preferably 1, 2, 3, 4, or 5additional amino acids, and/or 1, 2 or 3 substitutions, deletions,additions, or a mixture thereof. Preferably, the number ofsubstitutions, additions, deletions or a mixture thereof depends on thelength of the sequence. For instance, the percentage of substitutions,deletions, additions, or a mixture thereof may be no more than 30%,preferably no more than 25%.

As used herein, the term “substitution” refers to the exchange of asingle amino-acid by another in a peptide sequence.

As used herein, the term “deletion” refers to the removal of a singleamino-acid in a peptide sequence.

As used herein, the term “insertion” or “addition” are equivalent andrefer to the addition of a single amino-acid in a peptide sequence.

By “substitutions, additions, deletions” is intended a substitution,addition, deletion of one amino acid. Then, when it is referred to “1,2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, additions, deletions or amixture thereof”, “1, 2, 3, 4, or 5 substitutions, additions, deletionsor a mixture thereof” or “1, 2 or 3 substitutions, deletions, additions,or a mixture thereof”, it means respectively “1, 2, 3, 4, 5, 6, 7, 8, 9or 10 modification(s) of an amino acid selected from substitutions,additions, deletions and a mixture thereof”, “1, 2, 3, 4, or 5modification(s) of an amino acid selected from substitutions, additions,deletions or a mixture thereof” or “1, 2 or 3 modification(s) of anamino acid selected from substitutions, deletions, additions, or amixture thereof”. “1, 2, 3, 4, or 5 substitutions, additions, deletionsor a mixture thereof” also means “from 1 to 5 substitutions, additions,deletions or a mixture thereof”. “1, 2, or 3 substitutions, additions,deletions or a mixture thereof” also means “from 1 to 3 substitutions,additions, deletions or a mixture thereof”.

In the peptide sequences disclosed herein, the amino acids arerepresented by their one letter code according to the followingnomenclature: A: alanine; C: cysteine; D: aspartic acid; E: glutamicacid; F: phenylalanine; G: glycine; H: histidine; I: isoleucine; K:lysine; L: leucine; M: methionine; N: asparagine; P: proline; Q:glutamine; R: arginine; S: serine; T: threonine; V: valine; W:tryptophane and Y: tyrosine.

As used herein, the terms “sequence identity” or “identity” refers to anexact amino acid to amino acid correspondence of two peptides. Percentof identity can be determined by a direct comparison of the sequenceinformation between two molecules by aligning the sequences, countingthe exact number of matches between the two aligned sequences, dividingby the length of the shorter sequence, and multiplying the result by100.

The sequence identity can be determined by alignment of two peptidesequences using global or local alignment algorithms, depending on thelength of the two sequences. Sequences of similar lengths are preferablyaligned using global alignment algorithms (e.g. Needleman Wunsch) whichaligns the sequences optimally over the entire length, while sequencesof substantially different lengths are preferably aligned using a localalignment algorithm (e.g. Smith Waterman). Sequences may then bereferred to as “substantially identical” or “essentially similar” whenthey (when optimally aligned by for example the programs GAP or BESTFITusing default parameters) share at least a certain minimal percentage ofsequence identity. GAP uses the Needleman and Wunsch global alignmentalgorithm to align two sequences over their entire length (full length),maximizing the number of matches and minimizing the number of gaps. Aglobal alignment is suitably used to determine sequence identity whenthe two sequences have similar lengths.

By “increased”, “increase” or “enhance” is intended to refer to ameasurement increased by at least 10, 20, 30, 40, 50, 60, 70, 80 or 90%when compared to the measurement measured in absence of the testedmolecule in the same conditions. By “decreased” or “decrease” isintended to refer to a measurement decreased by at least 10, 20, 30, 40,50, 60, 70, 80 or 90% when compared to the measurement measured inabsence of the tested molecule in the same conditions.

As used herein, the term “treatment”, “treat” or “treating” refers toany act intended to ameliorate the health status of patients, such ascure, alleviate or delay of the disease. It includes preventive as wellas therapeutic treatment. For instance, it may refer to a delay or ablockade of the evolution from NAFLD to NASH, from NASH to NASH withfibrosis, from NASH to cirrhosis, from NASH or cirrhosis tohepatocellular carcinoma. The term treatment designates in particularthe correction, retardation, or reduction of the hepatic steatosis. Theterm “treatment” also designates an improvement in the liver steatosis,in liver inflammation, in liver fibrosis, in liver enzymes(aminotransferases such as AST and ALT), and/or in fatty liver index(Bedgni et al, BMC Gastroenterol. 2006 Nov. 2; 6:33). In particular, thetreatment lowers or decreases or delays the in the liver steatosis, inliver inflammation, in liver fibrosis, in liver enzymes(aminotransferases such as AST and ALT), and/or in fatty liver index. Inthe context of fibrosis, it may refer to a delay or a blockade of theevolution of fibrosis. In particular, the term treatment designates inparticular the correction, retardation, or reduction of fibrosis.

As used herein, the term “effective amount” refers to a quantity of apeptide of the present disclosure or of a pharmaceutical composition ofthe present disclosure which treats or delays the progression or onsetof nonalcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver(NAFL), non-alcoholic steatohepatitis (NASH), hepatic steatosis (fattyliver), liver fibrosis, liver inflammation, cirrhosis, or hepatocellularcarcinoma. It can also refer to a quantity of a peptide of the presentdisclosure or of a pharmaceutical composition of the present disclosurewhich treats or delays fibrosis.

As used herein, the terms “active principle”, “active ingredient” and“active pharmaceutical ingredient” are equivalent and refers to acomponent of a pharmaceutical composition having a therapeutic effect.

As used herein, the term “therapeutic effect” refers to an effectinduced by an active ingredient, such as a peptide of the presentdisclosure, or by a pharmaceutical composition according to the presentdisclosure, capable to treat or to delay the progression or onset of adisease such as nonalcoholic fatty liver disease (NAFLD), non-alcoholicfatty liver (NAFL), non-alcoholic steatohepatitis (NASH), hepaticsteatosis (fatty liver), liver fibrosis, liver inflammation, cirrhosis,hepatocellular carcinoma or fibrosis.

As used herein, the term “excipient or pharmaceutically acceptablecarrier” refers to any ingredient except active ingredients that ispresent in a pharmaceutical composition. Its addition may be aimed toconfer a particular consistency or other physical or gustativeproperties to the final product. An excipient or pharmaceuticallyacceptable carrier must be devoid of any interaction, in particularchemical, with the active ingredients.

As used herein, the terms “subject”, “individual” or “patient” areinterchangeable and refer to an animal, preferably to a mammal, evenmore preferably to a human, including adult, child, newborn and human atthe prenatal stage.

In the present document, the term “about” refers to a range of values of±10% of the specified value. For example, “about 50” comprise values of±10% of 50, i.e. values in the range between 45 and 55. Preferably, theterm “about” refers to a range of values of ±5% of the specified value.

As used herein, “nonalcoholic fatty liver disease” and “NAFLD” refer toa disease defined by the presence of macrovascular steatosis in thepresence of less than 20 g of alcohol ingestion per day. NAFLD is themost common liver disease in the United States, and is commonlyassociated with insulin resistance/type 2 diabetes mellitus and obesity.NAFLD is manifested by steatosis, steatohepatitis, cirrhosis, andsometimes hepatocellular carcinoma. For a review of NAFLD, see Tolmanand Dalpiaz (2007) Ther. Clin. Risk. Manag., 3(6): 1153-1163 the entirecontents of which are incorporated herein by reference.

As used herein, the terms “steatosis,” “hepatic steatosis,” and “fattyliver” refer to the accumulation of triglycerides and other fats in theliver cells.

As used herein, the term “Nonalcoholic steatohepatitis” or “NASH” refersto liver inflammation and damage caused by a buildup of fat in theliver. NASH is part of a group of conditions called nonalcoholic fattyliver disease (NAFLD). NASH resembles alcoholic liver disease, butoccurs in people who drink little or no alcohol. The major feature inNASH is fat in the liver, along with inflammation and damage. Mostpeople with NASH feel well and are not aware that they have a liverproblem. Nevertheless, NASH can be severe and can lead to cirrhosis, inwhich the liver is permanently damaged and scarred and no longer able towork properly. NASH is usually first suspected in a person who is foundto have elevations in liver tests that are included in routine bloodtest panels, such as alanine aminotransferase (ALT) or aspartateaminotransferase (AST). When further evaluation shows no apparent reasonfor liver disease (such as medications, viral hepatitis, or excessiveuse of alcohol) and when x rays or imaging studies of the liver showfat, NASH is suspected. The only means of proving a diagnosis of NASHand separating it from simple fatty liver is a liver biopsy.

As used herein, the term “cirrhosis,” defined histologically, is adiffuse hepatic process characterized by fibrosis and conversion of thenormal liver architecture into structurally abnormal nodules.

NAFLD may be differentiated from NASH by the NAFLD Activity Score (NAS),the sum of the histopathology scores of a liver biopsy for steatosis (0to 3), lobular inflammation (0 to 2), and hepatocellular ballooning (0to 2). A NAS of <3 corresponds to NAFLD, 3-4 corresponds to borderlineNASH, and >5 corresponds to NASH. The biopsy is also scored for fibrosis(0 to 4).

Peptides

The peptide(s) according to the present disclosure present(s) thefollowing features:

-   -   it does not simultaneously comprise one methionine, one proline        and one arginine;    -   preferably, it adopts a secondary structure which is a helix,        preferably an alpha helix;    -   it comprises, consists essentially in or consists in a sequence        from a segment of the kinase domain of a PKC (Protein Kinase C),        preferably a segment of at least 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 25 consecutive        residues of the kinase domain of a PKC (Protein Kinase C); and    -   the peptide sequence may comprise 1, 2, 3, 4, or 5 amino acid        modification(s) selected from substitution(s), deletion(s),        addition(s), and a mixture thereof within said sequence of a        segment of the kinase domain of the PKC.

The peptide(s) may further present one or several of the followingfeatures:

-   -   it has a length of less than 80 amino acids, more preferably        less than 60 amino acids, still preferably less than 40 amino        acids, and even more preferably less than 30 amino acids;    -   it has a length of at least 5 amino acids and less than 40 amino        acids, preferably a length of at least 5 amino acids and less        than 30 amino acids, more preferably of at least 5 amino acids        and less than 25 amino acids;    -   it is modified by a cross-link;    -   it is able to interfere with ALMS1-PKC interaction, in        particular to decrease or prevent the interaction between ALMS1        and αPKC; or it is not able to interfere with ALMS1-PKC        interaction, in particular to decrease or prevent the        interaction between ALMS1 and αPKC;    -   it modifies the expression levels of the FATPs expression in        adipose tissue, preferentially it decreases the FATP2 expression        in adipose tissue;    -   it decreases the liver steatosis, the amount of fat in liver,        the size of fat droplets in liver, and/or the fatty liver index;    -   it induces the expression levels of heme oxygenase 1 in the        adipocytes.

The peptide(s) may further present one or several of the followingfeatures:

-   -   it has a length of less than 80 amino acids, more preferably        less than 60 amino acids, still preferably less than 40 amino        acids, and even more preferably less than 30 amino acids;    -   it has a length of at least 5 amino acids and less than 40 amino        acids, preferably a length of at least 5 amino acids and less        than 30 amino acids, more preferably of at least 5 amino acids        and less than 25 amino acids;    -   it is modified by a cross-link;    -   it is not able to interfere with ALMS1-PKC interaction, in        particular to decrease or prevent the interaction between ALMS1        and αPKC;    -   it modifies the expression levels of the expression of collagen        IV and LOXL2 (Lysyl oxidase homolog 2), preferentially it        decreases the expression of collagen IV and LOXL2, in particular        the LOXL2 expression in liver and/or plasma;    -   it decreases the fibrosis;    -   It is capable of decreasing the lysophosphatidylcholine (LPC)        lipid content in the tissues and in circulation, preferentially        the 18:2 LPC.

In one aspect, the peptide of the present disclosure comprises, consistsessentially in or consists in a sequence from a segment of the kinasedomain of a PKC (Protein Kinase C). The PKC can be selected fromconventional PKC, novel PKC and atypical PKC. In particular, the PKC canbe selected from conventional PKC. Preferably, the PKC can be selectedfrom the group consisting of α, βI, βII, and γ PKCs. More preferably,the PKC can be selected from the group consisting of α, βI, and βIIPKCs. Even more preferably, the PKC is an α PKC, preferably a human αPKC, more preferably a human αPKC of SEQ ID NO: 1. The kinase domain ofthe human αPKC is disclosed in SEQ ID NO: 2.

The segment of the kinase domain of a PKC has at least 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 25 consecutiveresidues of the kinase domain of a PKC. In one aspect, the segment ofthe kinase domain of a PKC has from 5 to 40 consecutive residues of thekinase domain of a PKC (optionally, from 5 to 30 or from 5 to 25 or from7 to 25 or from 8 to 25 or from 9 to 25 or from 10 to 25 or from 11 to25 or from 12 to 25).

The kinase domain of PKC from which the segment is selected haspreferably at least 40% of identity with the sequence of SEQ ID NO: 2,more preferably at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95%of identity with the sequence of SEQ ID NO: 2.

Preferably, said sequence of a segment of the kinase domain of a PKCcorresponds to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 99% of the sequence of the peptide. In a particular embodiment, thepeptide sequence according to the present disclosure consist in thesequence of a segment of SEQ ID NO: 1.

When the segment of the kinase domain of a PKC comprises one methionineand/or one proline and/or one arginine, then the sequence can bemodified (i.e., by introducing substitution(s)) so as to remove all theproline residues, and/or all the methionine residues, and/or all thearginine residues. For instance, the sequence can be modified (i.e., byintroducing substitution(s)) so as to remove all the proline residues.Alternatively, the sequence can be modified (i.e., by introducingsubstitution(s)) so as to remove all the methionine residues. Otherwise,the sequence can be modified (i.e., by introducing substitution(s)) soas to remove all the arginine residues. In one aspect, the sequence canbe modified (i.e., by introducing substitution(s)) so as to remove allthe proline and methionine residues. In another aspect, the sequence canbe modified (i.e., by introducing substitution(s)) so as to remove allthe proline and arginine residues. In an additional aspect, the sequencecan be modified (i.e., by introducing substitution(s)) so as to removeall the methionine and arginine residues. More preferably, the sequencecan be modified (i.e., by introducing substitution(s)) so as to removeall the proline residues, all the methionine residues, and all thearginine residues.

Preferably, the peptide comprises no more than 20, preferably no morethan 15, more preferably no more than 10, amino acid modificationsselected from substitutions, deletions, additions, and a mixturethereof. In a particularly preferred embodiment, the peptide maycomprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modificationsselected from substitution(s), deletion(s), addition(s), and a mixturethereof, preferably 1, 2, 3, 4, or 5, more preferably 1, 2 or 3.

For instance, the peptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%of identity with the sequence of a segment of the kinase domain PKC,preferably of SEQ ID NO: 2. In one embodiment, the part of the sequenceof the peptide corresponding to SEQ ID NO: 2 has at least 70%, 75%, 80%,85%, 90%, 95%, of identity with the sequence of a segment of SEQ ID NO:2.

For instance, the sequence of a segment of the kinase domain of the PKCmay belong to the sequences between positions 339 and 432 of SEQ ID NO:1, between positions 434 and 544 of SEQ ID NO: 1, between positions 546and 561 of SEQ ID NO: 1, between positions 563 and 565 of SEQ ID NO: 1,or between positions 568 and 595 of SEQ ID NO: 1.

In one embodiment, the sequence of a segment of the kinase domain of PKCmay not include the following residues: G433, E545, 5562, 5566 of SEQ IDNO: 1.

In one aspect, the peptide of the present disclosure has an alpha helixstructure. As used herein, the terms “alpha helix” “α-helix”, “classicPauling-Corey-Branson α-helix” and “3.613-helix” are equivalent andrefer to each other. The term “alpha helix” refers to a common motif inthe secondary structure of proteins which is a right hand-coiled orspiral conformation (helix) in which every backbone N—H group donates ahydrogen bond to the backbone C═O group of the amino acid located threeor four residues earlier along the protein sequence. An alpha helix hasan average number of residues per helical turn of about 3.6 residues and13 atoms are involved in the ring formed by the hydrogen bond.

In a particular embodiment, the peptide of the present disclosure has analpha helix structure and/or has a sequence which is predictive of analpha helix structure. Methods to determine the structure of a peptideare well known from the man skilled in the art, such as CircularDichroism or NMR. Likewise, methods to predict an alpha helix structureof a peptide are well known from the man skilled in the art such asSTRIDE (Frishman D., Argos P., Proteins, vol. 23, no 4, 1995, p.566-579); DEFINE (Richards F. M., Kundrot C. E., Proteins, vol. 3, no 2,1988, p. 71-84); DSSP (Touw et al. Nucleic Acids Research 2015; 43:D364-D368; Kabsch & Sander. Biopolymers. 1983, 22, 2577-2637).

The alpha helices are located in the kinase domain at the followinglocations: 372-377; 381-392; 425-432; 437-456; 466-468; 502-504;507-510; 518-533; 543-552; 563-572; 577-579; 587-593 and 595-597 of SEQID NO: 1.

According, the peptide may comprise, consist essentially in or consistin at least one of the following sequences:

(SEQ ID NO: 3) VECTMVEKRVLA; (SEQ ID NO: 4) LMYHIQQV; LDN; PDY;(SEQ ID NO: 5) PEII; (SEQ ID NO: 6) SVDWWAYGVLLYEMLA;  (SEQ ID NO: 7)EDEDELFQSIME; PAK; (SEQ ID NO: 8) GERDVRE; AFF.

In a particular embodiment, the peptide may comprise, consistessentially in or consist in at least one of the following sequences:

(SEQ ID NO: 3) VECTMVEKRVLA; and (SEQ ID NO: 8) GERDVRE.

Optionally, the peptide may comprise, consist essentially in or consistin at least one of the following sequences: VECTMVEKRVLA (SEQ ID NO: 3);VECTXVEKRVLA (SEQ ID NO: 9); VECTMVEKXVLA (SEQ ID NO: 10); VECTXVEKXVLA(SEQ ID NO: 11); LMYHIQQV (SEQ ID NO: 4); LXYHIQQV (SEQ ID NO: 12); LDN;SVDWWAYGVLLYEMLA (SEQ ID NO: 6); SVDWWAYGVLLYEXLA (SEQ ID NO: 13);EDEDELFQSIME (SEQ ID NO: 7); EDEDELFQSIXE (SEQ ID NO: 14); GERDVRE (SEQID NO: 8); GEXDVRE (SEQ ID NO: 15); GERDVXE (SEQ ID NO: 16); GEXDVXE(SEQ ID NO: 17); LDN; AFF; PDY; XDY; PEII (SEQ ID NO: 5); XEII (SEQ IDNO: 18); PAK; XAK; wherein X is any amino acid except M, P and R.Preferably, X an amino acid favorable to an α-helix secondary structure.For instance, X may be selected from the group consisting of A, D, N, C,G, Q, E, H, L, K, F, S, W and Y, more preferably A, D, N, G, Q, E, H, L,K, F, S, W and Y. In one aspect, the peptide may comprise, consistessentially in or consist in at least one of the following sequences:VECTMVEKRVLA (SEQ ID NO: 3); VECTXVEKRVLA (SEQ ID NO: 9); VECTMVEKXVLA(SEQ ID NO: 10); VECTXVEKXVLA (SEQ ID NO: 11); LMYHIQQV (SEQ ID NO: 4);LXYHIQQV (SEQ ID NO: 12); SVDWWAYGVLLYEMLA (SEQ ID NO: 6);SVDWWAYGVLLYEXLA (SEQ ID NO: 13); EDEDELFQSIME (SEQ ID NO: 7);EDEDELFQSIXE (SEQ ID NO: 14); GERDVRE (SEQ ID NO: 8); GEXDVRE (SEQ IDNO: 15); GERDVXE (SEQ ID NO: 16); GEXDVXE (SEQ ID NO: 17); wherein X isany amino acid except M, P and R. In particular, the peptide maycomprise, consist essentially in or consist in at least one of thefollowing sequences: VECTMVEKRVLA (SEQ ID NO: 3); VECTXVEKRVLA (SEQ IDNO: 9); VECTMVEKXVLA (SEQ ID NO: 10); VECTXVEKXVLA (SEQ ID NO: 11);LXYHIQQV (SEQ ID NO: 12); SVDWWAYGVLLYEXLA (SEQ ID NO: 13); EDEDELFQSIXE(SEQ ID NO: 14); GERDVRE (SEQ ID NO: 8); GEXDVRE (SEQ ID NO: 15);GERDVXE (SEQ ID NO: 16); GEXDVXE (SEQ ID NO: 17); wherein X is any aminoacid except M, P and R. For instance, the peptide may comprise at leastone of the following sequences:

(SEQ ID NO: 3)  VECTMVEKRVLA or   (SEQ ID NO: 19) VECTTVEKEVLA.

Optionally, the peptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10substitution(s), deletion(s), addition(s), or a mixture thereof,preferably, 1, 2, 3, 4, or 5 substitution(s), deletion(s), addition(s),or a mixture thereof, more preferably, 1, 2, or 3 substitution(s).

Optionally, the peptide may comprise, consist essentially in or consistin at least one of the following sequences: VECTMVEKRVLA (SEQ ID NO: 3)with optionally 1, 2, 3, 4, or 5 modification(s) of an amino acidselected from substitution(s), deletion(s), addition(s), and a mixturethereof, more preferably, 1, 2, or 3 modification(s) of an amino acidselected from substitution(s) deletion(s), addition(s), and a mixturethereof; VECTXVEKRVLA (SEQ ID NO: 9) with optionally modification(s) ofan amino acid selected from 1, 2, 3, 4, or 5 modification(s) of an aminoacid selected from substitution(s), deletion(s), addition(s), and amixture thereof, more preferably, 1, 2, or 3 modification(s) of an aminoacid selected from substitution(s) deletion(s), addition(s), and amixture thereof; VECTMVEKXVLA (SEQ ID NO: 10) with optionally 1, 2, 3,4, or 5 modification(s) of an amino acid selected from substitution(s),deletion(s), addition(s), and a mixture thereof, more preferably, 1, 2,or 3 modification(s) of an amino acid selected from substitution(s)deletion(s), addition(s), and a mixture thereof; VECTXVEKXVLA (SEQ IDNO: 11) with optionally 1, 2, 3, 4, or 5 modification(s) of an aminoacid selected from substitution(s), deletion(s), addition(s), and amixture thereof, more preferably, 1, 2, or 3 modification(s) of an aminoacid selected from substitution(s) deletion(s), addition(s), and amixture thereof; LMYHIQQV (SEQ ID NO: 4) with optionally 1, 2, or 3modification(s) of an amino acid selected from substitution(s)deletion(s), addition(s), and a mixture thereof; LXYHIQQV (SEQ ID NO:12) with optionally 1, 2, or 3 modification(s) of an amino acid selectedfrom substitution(s) deletion(s), addition(s), and a mixture thereof;LDN; SVDWWAYGVLLYEMLA (SEQ ID NO: 6) with optionally 1, 2, 3, 4, or 5modification(s) of an amino acid selected from substitution(s),deletion(s), addition(s), and a mixture thereof, more preferably, 1, 2,or 3 modification(s) of an amino acid selected from substitution(s)deletion(s), addition(s), and a mixture thereof; SVDWWAYGVLLYEXLA (SEQID NO: 13) with optionally 1, 2, 3, 4, or 5 modification(s) of an aminoacid selected from substitution(s), deletion(s), addition(s), and amixture thereof, more preferably, 1, 2, or 3 modification(s) of an aminoacid selected from substitution(s) deletion(s), addition(s), and amixture thereof; EDEDELFQSIME (SEQ ID NO: 7) with optionally 1, 2, 3, 4,or 5 modification(s) of an amino acid selected from substitution(s),deletion(s), addition(s), and a mixture thereof, more preferably, 1, 2,or 3 modification(s) of an amino acid selected from substitution(s)deletion(s), addition(s), and a mixture thereof; EDEDELFQSIXE (SEQ IDNO: 14) with optionally 1, 2, 3, 4, or 5 modification(s) of an aminoacid selected from substitution(s), deletion(s), addition(s), and amixture thereof, more preferably, 1, 2, or 3 modification(s) of an aminoacid selected from substitution(s) deletion(s), addition(s), and amixture thereof; GERDVRE (SEQ ID NO: 8) with optionally 1, 2, or 3modification(s) of an amino acid selected from substitution(s)deletion(s), addition(s), and a mixture thereof; GEXDVRE (SEQ ID NO: 15)with optionally 1, 2, or 3 modification(s) of an amino acid selectedfrom substitution(s) deletion(s), addition(s), and a mixture thereof;GERDVXE (SEQ ID NO: 16) with optionally 1, 2, or 3 modification(s) of anamino acid selected from substitution(s) deletion(s), addition(s), and amixture thereof; GEXDVXE (SEQ ID NO: 17) with optionally 1, 2, or 3modification(s) of an amino acid selected from substitution(s)deletion(s), addition(s), and a mixture thereof; LDN; AFF; PDY; XDY;PEII (SEQ ID NO: 5); XEII (SEQ ID NO: 18); PAK; XAK; wherein X is anyamino acid except M, P and R. Preferably, X an amino acid favorable toan α-helix secondary structure. For instance, X may be selected from thegroup consisting of A, D, N, C, G, Q, E, H, L, K, F, S, W and Y, morepreferably A, D, N, G, Q, E, H, L, K, F, S, W and Y.

In one aspect, the peptide may comprise, consist essentially in orconsist in at least one of the following sequences: VECTMVEKRVLA (SEQ IDNO: 3) with optionally 1, 2, or 3 modification(s) of an amino acidselected from substitution(s) deletion(s), addition(s), and a mixturethereof; VECTXVEKRVLA (SEQ ID NO: 9) with optionally modification(s) ofan amino acid selected from 1, 2, or 3 modification(s) of an amino acidselected from substitution(s) deletion(s), addition(s), and a mixturethereof; VECTMVEKXVLA (SEQ ID NO: 10) with optionally 1, 2, or 3modification(s) of an amino acid selected from substitution(s)deletion(s), addition(s), and a mixture thereof; VECTXVEKXVLA (SEQ IDNO: 11) with optionally 1, 2, or 3 modification(s) of an amino acidselected from substitution(s) deletion(s), addition(s), and a mixturethereof; LMYHIQQV (SEQ ID NO: 4) with optionally 1, 2, or 3modification(s) of an amino acid selected from substitution(s)deletion(s), addition(s), and a mixture thereof; LXYHIQQV (SEQ ID NO:12) with optionally 1, 2, or 3 modification(s) of an amino acid selectedfrom substitution(s) deletion(s), addition(s), and a mixture thereof;SVDWWAYGVLLYEMLA (SEQ ID NO: 6) with optionally 1, 2, or 3modification(s) of an amino acid selected from substitution(s)deletion(s), addition(s), and a mixture thereof; SVDWWAYGVLLYEXLA (SEQID NO: 13) with optionally 1, 2, or 3 modification(s) of an amino acidselected from substitution(s) deletion(s), addition(s), and a mixturethereof; EDEDELFQSIME (SEQ ID NO: 7) with optionally 1, 2, or 3modification(s) of an amino acid selected from substitution(s)deletion(s), addition(s), and a mixture thereof; EDEDELFQSIXE (SEQ IDNO: 14) with optionally 1, 2, or 3 modification(s) of an amino acidselected from substitution(s) deletion(s), addition(s), and a mixturethereof; GERDVRE (SEQ ID NO: 8) with optionally 1, 2, or 3modification(s) of an amino acid selected from substitution(s)deletion(s), addition(s), and a mixture thereof; GEXDVRE (SEQ ID NO: 15)with optionally 1, 2, or 3 modification(s) of an amino acid selectedfrom substitution(s) deletion(s), addition(s), and a mixture thereof;GERDVXE (SEQ ID NO: 16) with optionally 1, 2, or 3 modification(s) of anamino acid selected from substitution(s) deletion(s), addition(s), and amixture thereof; GEXDVXE (SEQ ID NO: 17) with optionally 1, 2, or 3modification(s) of an amino acid selected from substitution(s)deletion(s), addition(s), and a mixture thereof; wherein X is any aminoacid except M, P and R. Preferably, X an amino acid favorable to anα-helix secondary structure. For instance, X may be selected from thegroup consisting of A, D, N, C, G, Q, E, H, L, K, F, S, W and Y, morepreferably A, D, N, G, Q, E, H, L, K, F, S, W and Y.

In particular, the peptide may comprise, consist essentially in orconsist in at least one of the following sequences: VECTMVEKRVLA (SEQ IDNO: 3) with optionally 1, 2, or 3 modification(s) of an amino acidselected from substitution(s) deletion(s), addition(s), and a mixturethereof; VECTXVEKRVLA (SEQ ID NO: 9) with optionally modification(s) ofan amino acid selected from 1, 2, or 3 modification(s) of an amino acidselected from substitution(s) deletion(s), addition(s), and a mixturethereof; VECTMVEKXVLA (SEQ ID NO: 10) with optionally 1, 2, or 3modification(s) of an amino acid selected from substitution(s)deletion(s), addition(s), and a mixture thereof; VECTXVEKXVLA (SEQ IDNO: 11) with optionally 1, 2, or 3 modification(s) of an amino acidselected from substitution(s) deletion(s), addition(s), and a mixturethereof; GERDVRE (SEQ ID NO: 8) with optionally 1, 2, or 3modification(s) of an amino acid selected from substitution(s)deletion(s), addition(s), and a mixture thereof; GEXDVRE (SEQ ID NO: 15)with optionally 1, 2, or 3 modification(s) of an amino acid selectedfrom substitution(s) deletion(s), addition(s), and a mixture thereof;GERDVXE (SEQ ID NO: 16) with optionally 1, 2, or 3 modification(s) of anamino acid selected from substitution(s) deletion(s), addition(s), and amixture thereof; GEXDVXE (SEQ ID NO: 17) with optionally 1, 2, or 3modification(s) of an amino acid selected from substitution(s)deletion(s), addition(s), and a mixture thereof; wherein X is any aminoacid except M, P and R. Preferably, X an amino acid favorable to anα-helix secondary structure. For instance, X may be selected from thegroup consisting of A, D, N, C, G, Q, E, H, L, K, F, S, W and Y, morepreferably A, D, N, G, Q, E, H, L, K, F, S, W and Y.

For instance, the peptide may comprise at least one of the followingsequences:

(SEQ ID NO: 19) VECTMVEKRVLA or VECTTVEKEVLA.

In one aspect, the peptide may comprise, consist essentially in orconsist in at least one of the following sequences:

-   -   a) VECTXVEKXVLALLDKXXFLTQLHS (SEQ ID NO: 20) wherein X is any        amino acid except M, P and R, preferably, an amino acid        favorable to an α-helix secondary structure, more preferably        selected from the group consisting of A, D, N, C, G, Q, E, H, L,        K, F, S, W and Y, still more preferably A, D, N, G, Q, E, H, L,        K, F, S, W and Y, with optionally 1, 2, 3, 4, or 5        modification(s) of an amino acid selected from substitution(s),        deletion(s), addition(s), and a mixture thereof, more        preferably, 1, 2, or 3 modification(s) of an amino acid selected        from substitution(s) deletion(s), addition(s), and a mixture        thereof;    -   b) VECTMVEKRVLALLDKXXFLTQLHS (SEQ ID NO: 21) wherein X is any        amino acid except M, P and R, preferably, an amino acid        favorable to an α-helix secondary structure, more preferably        selected from the group consisting of A, D, N, C, G, Q, E, H, L,        K, F, S, W and Y, still more preferably A, D, N, G, Q, E, H, L,        K, F, S, W and Y, with optionally 1, 2, 3, 4, or 5        modification(s) of an amino acid selected from substitution(s),        deletion(s), addition(s), and a mixture thereof, more        preferably, 1, 2, or 3 modification(s) of an amino acid selected        from substitution(s) deletion(s), addition(s), and a mixture        thereof;    -   c) VECTXVEKRVLALLDKPPFLTQLHS (SEQ ID NO: 22) wherein X is any        amino acid except M, P and R, preferably, an amino acid        favorable to an α-helix secondary structure, more preferably        selected from the group consisting of A, D, N, C, G, Q, E, H, L,        K, F, S, W and Y, still more preferably A, D, N, G, Q, E, H, L,        K, F, S, W and Y, with optionally 1, 2, 3, 4, or 5        modification(s) of an amino acid selected from substitution(s),        deletion(s), addition(s), and a mixture thereof, more        preferably, 1, 2, or 3 modification(s) of an amino acid selected        from substitution(s) deletion(s), addition(s), and a mixture        thereof;    -   d) VECTMVEKXVLALLDKPPFLTQLHS (SEQ ID NO: 23) wherein X is any        amino acid except M, P and R, preferably, an amino acid        favorable to an α-helix secondary structure, more preferably        selected from the group consisting of A, D, N, C, G, Q, E, H, L,        K, F, S, W and Y, still more preferably A, D, N, G, Q, E, H, L,        K, F, S, W and Y, with optionally 1, 2, 3, 4, or 5        modification(s) of an amino acid selected from substitution(s),        deletion(s), addition(s), and a mixture thereof, more        preferably, 1, 2, or 3 modification(s) of an amino acid selected        from substitution(s) deletion(s), addition(s), and a mixture        thereof;        and the sequence of any segment of at least 5, 6, 7, 8, 9, 10,        11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 25        consecutive residues of any sequence a) to d).

In another particular embodiment, the peptide according to the presentdisclosure is designed or modified in order to maintain it in an alphahelical conformation. As known in the art, this can be achieved via avariety of methods, including modification of the amino acid sequencewith substitution of amino acids not critical for biological effects,use of non-natural amino acids, peptide cyclization, and modificationsto the peptide backbone or addition of chemical links between aminoacids in the peptide chain. Such modifications can be made to peptides,for example, to increase their thermal and protease stability.

In particular, the peptide of the present disclosure is modified by achemical cross-link. For instance, the peptide can be a stapled peptide.In one embodiment, the peptide of the present disclosure is stapled. Theterm “stapled peptide” or “stitched peptide”, as used herein, refers toan artificially modified peptide in which the peptide secondarystructure is stabilized with one or more artificial molecular crosslinks(bridges) that connect adjacent turns of α-helices in the peptide. Themethods for preparing stapled peptides are well known in the art, forinstance in Verdine & Hilinski (2012, Methods Enzymol, 503, 3-33),WO10033617 and WO10011313, the disclosure of which is incorporatedherein by reference.

In one embodiment, the crosslinks of the stapled peptide of the presentdisclosure are i+3, and/or i+4, and/or i+7 crosslinks. In a peptide, a“i+3 crosslink” is a crosslink between an amino acid, the “i” aminoacid, and another amino acid present at a distance of 3 amino acidresidues from the i amino acid. In a peptide, a “i+4 crosslink” is acrosslink between an amino acid, the “i” amino acid, and another aminoacid present at a distance of 4 amino acid residues from the i aminoacid. In a peptide, a “i+7 crosslink” is a crosslink between an aminoacid, the “i” amino acid, and another amino acid present at a distanceof 7 amino acid residues from the i amino acid.

For the shortest sequences, in particular those including three to fourresidues, the cross-link is i+3 and i+4 and it is introduced betweenresidues which are outside of this sequence. When the sequences are longenough, the cross-link of i+7 is preferred.

To illustrate this aspect on one particular peptide, the peptide maycomprise, consist essentially in or consist in one of the followingsequences:

(SEQ ID NO: 24) VECTM X EKRVLA X (SEQ ID NO: 25) VECTX X EKRVLA X(SEQ ID NO: 26) VECTM X EKXVLA X (SEQ ID NO: 27) VECTX X EKXVLA X(SEQ ID NO: 28) VECTXXEKXVLAXLDKXXFLTQLHS (SEQ ID NO: 29) VECTM X EKRVLAX LDKXXFLTQLHS (SEQ ID NO: 30) VECTX X EKRVLA X LDKPPFLTQLHS(SEQ ID NO: 31) VECTM X EKXVLA X LDKPPFLTQLHS

-   -   wherein the residues which are bold and underlined X carry the        stapling and is any amino acid derivative suitable for stapling;        and    -   wherein X is any amino acid except M, P and R, preferably, an        amino acid favorable to an α-helix secondary structure, more        preferably selected from the group consisting of A, D, N, C, G,        Q, E, H, L, K, F, S, W and Y, still more preferably A, D, N, G,        Q, E, H, L, K, F, S, W and Y, and    -   with the sequence having optionally 1, 2, 3, 4, or 5        modification(s) of an amino acid selected from substitution(s),        deletion(s), addition(s), and a mixture thereof, more        preferably, 1, 2, or 3 modification(s) of an amino acid selected        from substitution(s) deletion(s), addition(s), and a mixture        thereof.

For instance, in the context of a i+7 stapling, the first X is a2-(7-octenyl)amino acid (for instance a 2-(7-octenyl)alanine or a2-(7-octenyl)arginine) and the second X is a 2-(4-pentenyl)amino acid(for instance a 2-(4-pentenyl)alanine or a 2-(4-pentenyl)serine).Specific combinations can 2-(7-octenyl)alanine and2-(4-pentenyl)alanine; 2-(7-octenyl)alanine and 2-(4-pentenyl)serine;2-(7-octenyl)arginine and 2-(4-pentenyl)alanine; or2-(7-octenyl)arginine and 2-(4-pentenyl)serine.

In a particular embodiment, the peptide can be

(SEQ ID NO: 32) VECTT R EKEVLA S LDKAAFLTQLHS

-   -   wherein R and S carry the stapling, being preferably        2-(7-octenyl)arginine and 2-(4-pentenyl)serine, respectively;    -   with the sequence having optionally 1, 2, 3, 4, or 5        modification(s) of an amino acid selected from substitution(s),        deletion(s), addition(s), and a mixture thereof, more        preferably, 1, 2, or 3 modification(s) of an amino acid selected        from substitution(s) deletion(s), addition(s), and a mixture        thereof.

In a particular embodiment, the peptide according to the presentdisclosure is a cyclic peptide. As used herein, the term “cyclicpeptide” or “circular peptide” are equivalent and refers to a peptide inwhich the N-terminus and the C-terminus, or the N-terminus and the sidechain of another amino acid, preferably the C-terminal amino acid, orthe C-terminus and the side chain of another amino acid, preferably theN-terminal amino acid, or the side chain of an amino acid and the sidechain of another amino acid, preferably the N-terminal amino acid andthe C-terminal amino acid, are linked with a covalent bond thatgenerates a ring structure. As used herein, the term “N-terminus”,“amino-terminus”, “NH2-terminus”, “N-terminal end” and “amine-terminus”are equivalent and refer to the free amine group (—NH2) present on thefirst amino acid of the peptide. As used herein, the term “C-terminus”,“carboxyl-terminus”, “carboxy-terminus”, “C-terminal end”, and“COOH-terminus” are equivalent and refer to the free carboxyl group(—COOH) present on the last amino acid of the peptide.

In one embodiment, the peptide according to the present disclosure has alength of less than 80 amino acids, more preferably less than 60 aminoacids, still preferably less than 40 amino acids, and even morepreferably less than 30 amino acids. In a particular embodiment, thepeptide according to the present disclosure has a length of less than 25amino acids. In another particular embodiment, the peptide according tothe present disclosure has a length of less than 20 amino acids,preferably of less than 15 amino acids. Preferably, the peptide has aminimum length greater than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 amino acids. For instance, the peptide has alength of at least 4 amino acids and less than 40 amino acids,preferably a length of at least 4 amino acids and less than 30 aminoacids; more preferably of at least 6 amino acids and less than 25 aminoacids.

In one embodiment, the peptide according to the present disclosure iscapable of interfering with ALMS1-PKC interaction, in particular todecrease or prevent the interaction between ALMS1 and αPKC. In otherwords, the peptide according to the present disclosure is capable ofblocking the interaction between ALMS1 and αPKC. Alternatively, thepeptide according to the present disclosure is not capable ofinterfering with ALMS1-PKC interaction, in particular to decrease orprevent the interaction between ALMS1 and αPKC. In other words, thepeptide according to the present disclosure is not capable of blockingthe interaction between ALMS1 and αPKC.

In order to determine the effect of a peptide on the binding of αPKC toALMS1, any technology known by the person skilled in the art can becarried out, in particular any method suitable for determining proteininteractions. For example, recombinant or purified native ALMS1 or αPKCcan be bound to a surface plasmon resonance chip and the other moleculeflowed over the chip to assess the binding affinity, for example in aBiacore (General Electric, USA) machine.

The effect of peptide(s) on the binding of αPKC to ALMS1 is determiningby measuring the binding of αPKC to ALMS1 in absence and in presence ofthe tested peptide(s) and by comparing the bindings of αPKC to ALMS1.

In particular, immunoprecipitation assay using ALMS1 as bait can becarried. The assay can be carried out with cells, in particularadipocytes, cultured in absence and/or presence of insulin, preferablyin absence of insulin. The peptides to be tested are added in theculture medium. Then, αPKC is immunodetected.

By “decreased”, “decrease” or “prevent” is intended to refer to abinding decreased by at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% whencompared to the binding measured in absence of the tested molecule inthe same conditions.

In one embodiment, the peptide according to the present disclosure iscapable of decreasing the expression of FATP2 in adipose tissue.

FATP2 is also called Solute Carrier Family 27 Member 2 (SLC27A2). Thisprotein is disclosed in the database UniProtKB under 014975. The gene isdescribed in UniGene database under Hs.11729. Sequences of reference canbe found in NCBI under NP_003636.2 and NM_003645.3 for the isoform 1 andunder NP_001153101.1 and NM_001159629.1 for the isoform 2.

By “decreased” or “decrease” is intended to refer to an expressiondecreased by at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% whencompared to the expression measured in absence of the peptide in thesame conditions. The expression can be measured either at the proteinlevel (e.g., with antibodies) or at the mRNA level.

The expression can be measured at the protein level by any availablemethod such as immuno-histochemistry, semi-quantitative Western-blot orby protein or antibody arrays. Antibodies directed to FATP2 arecommercially available, for instance from Origene, ref TA350424 orTA333990; or Santa Cruz Biotechnology, ref sc-393906.

The expression can also be measured at the mRNA level by any availablemethod. Preferably, the expression level of FATP2 is determined bymeasuring the quantity of the mRNA transcripts by quantitative RT-PCR,real time quantitative RT-PCR, Nanostring technology PCR or byhigh-throughput sequencing technology such as RNA-Seq or sequencingtechnologies using microfluidic systems. More specifically, theexpression is measured by the method specified in the Example section.

In a particular embodiment, the effect on the FATP2 expression caused bythe peptide in the adipose tissue is preferably specific to FATP2. Inthis embodiment, the peptide can have no or less effect on theexpression of the other FATPs, i.e. FATP1, FATP3, FATP4, FATP5 andFATP6, in the adipose tissue, in particular of a mammal.

In one embodiment, the peptide according to the present disclosure iscapable of decreases the liver steatosis, the amount of fat in liver,the size of fat droplets in liver, and/or the fatty liver index.

In a particular embodiment, the peptide according to the presentdisclosure presents the following features

-   -   it does not simultaneously comprise one methionine, one proline        and one arginine;    -   it adopts a secondary structure which is a helix, preferably an        alpha helix; and    -   it comprises, consists essentially in or consists in a sequence        from a segment of the kinase domain of a PKC (Protein Kinase C);        -   and further present one, two, three, four or all following            features:    -   it modifies the expression levels of the FATPs expression in        adipose tissue, preferentially it decreases the FATP2 expression        in adipose tissue;    -   it decreases the liver steatosis, the amount of fat in liver,        the size of fat droplets in liver, and/or the fatty liver index;    -   it has a length of at least 4 amino acids and less than 40 amino        acids, preferably a length of at least 4 amino acids and less        than 30 amino acids, more preferably of at least 4 amino acids        and less than 25 amino acids;    -   it adopts a secondary structure which is a helix, preferably an        alpha helix;    -   it is modified by a cross-link.

In a more specific embodiment, the peptide according to the presentdisclosure presents the following features:

-   -   it decreases the liver steatosis, the amount of fat in liver,        the size of fat droplets in liver, and/or the fatty liver index;    -   it does not simultaneously comprise one methionine, one proline        and one arginine;    -   it has a length of at least 4 amino acids and less than 40 amino        acids, preferably a length of at least 4 amino acids and less        than 30 amino acids, more preferably of at least 4 amino acids        and less than 25 amino acids;    -   it adopts a secondary structure which is a helix, preferably an        alpha helix.

In another more specific embodiment, the peptide according to thepresent disclosure presents the following features:

-   -   it decreases the FATP2 expression in adipose tissue;    -   it does not simultaneously comprise one methionine, one proline        and one arginine;    -   it has a length of at least 4 amino acids and less than 40 amino        acids, preferably a length of at least 4 amino acids and less        than 30 amino acids, more preferably of at least 4 amino acids        and less than 25 amino acids;    -   it adopts a secondary structure which is a helix, preferably an        alpha helix.

In one embodiment, the peptide according to the present disclosure iscapable of decreasing the expression of collagen, in particular collagenIV, and LOXL2. Preferentially it decreases the expression of LOXL2, inparticular the LOXL2 expression in liver and/or plasma.

It can be capable of decreasing the lysophosphatidylcholine (LPC) lipidcontent in the tissues and in circulation, preferentially the 18:2 LPC.

The peptide according to the present disclosure may further comprise amoiety facilitating its cellular uptake or entry, in particular a PTD(protein transduction domain). PTD generally comprises a certain aminoacid sequence of 10 to 20 amino acids (Matsushita and Matsui, (2005), JMol Med 83, 324-328; Vivès et al, Biochimic et Biophysica Acta, 2008,1786, 126-138). PTD is mainly composed of basic amino acids such asarginine or lysine, and representative examples of the PTD includearginine rich peptides such as poly R₈ (RRRRRRRR (SEQ ID NO: 33)) or(RRPRRPRRPRRPRRP (SEQ ID NO: 34)), antennapedia or penetratin peptidesuch as (RQIKIWFQNRRMKWKK (SEQ ID NO: 35)) or HIV-Tat (YGRKKRRQRRR (SEQID NO: 36)).

The peptide according to the present disclosure can be made of naturalamino acids and/or unnatural amino acids. The term “unnatural aminoacids” is defined as an analog or derivative of a natural amino acid(i.e., Alanine, Valine, Glycine, Leucine, Isoleucine, Lysine, Arginine,Glutamic acid, Glutamine, Aspartic acid, Asparagine, Histidine,Tyrosine, Phenylalanine, Tryptophan, Serine, Proline, Threonine,Cysteine, Methionine). They present a modified side chain, e.g. shorter,longer or with different functional groups. Isomers D and L arecontemplated, in particular because isomers D are not sensible toproteases. In addition, modifications in some or all peptide bounds arealso contemplated in order to increase the proteolysis resistance, inparticular by (—CO—NH—) by (—CH₂—NH—), (—NH—CO—), (—CH₂—O—), (—CH₂—S—),(—CH₂—CH₂—), (—CO—CH₂—), (—CHOH—CH₂—), (—N═N—), and/or (—CH═CH—). Thepeptide can present a carboxylic C terminal end (—COO⁻) and an amide one(—CONH₂). The peptide can also be D-retro-inverso sequence of a peptideas disclosed herein. The N terminal can be modified, especially with anacetyl radical.

Optionally, the peptide can be PEGylated in order to increase itsstability. Further optionally the peptide can be formulated innon-aqueous protic solvent solutions such as propylene glycol andpolyethylene glycol. The peptide may also be packaged into poly lacticco-glycolic acid microsphere depot formulation. Many sustained-releasedelivery systems exist, and many of these are appropriate for use in thepresent disclosure. For example, polymer-based slow-release compositionsbased upon degradable polymers such as PLGA, poly-lactate orpoly-glycolate are suitable, as are lipid-based depot compositions, suchas those described in WO2005/117830 and/or WO2006/075124, the completedisclosures of which are being hereby incorporated by reference. Theformulation of active agents into biodegradable polymer depotformulations is well established and well known in the art, and thepeptides of the present disclosure may thus be formulated with theseusing known methods. Preferably, the composition of the presentdisclosure is capable of releasing the peptide at a functionalconcentration for at least 1 month.

In an additional aspect, the peptide according to the present disclosuredecreases the phenomenon of steatosis and any hepatic disorderassociated with NAFLD or NASH in the liver. The phenomenon of steatosisin the liver can be assessed by any method known from the man skilled inthe art. In particular, it is assessed by the method described in theexample section. For instance, the steatosis can be measured by imagingor biopsy. Peptides that decrease the phenomenon of steatosis in theliver can be conveniently screened for using any technology known in theart. In particular, a method for assessing the steatosis in the livercan comprise any method suitable for measuring the fat in the liver, thesize of the lipid droplets in the liver and/or measuring the fatty liverindex (Bedgni et al, BMC Gastroenterol. 2006 Nov. 2; 6:33).

By “a peptide” is intended to refer to a peptide as disclosed above or acombination of different peptides as disclosed above. For instance, 2,3, 4, 5 or 6 different peptides can be used, preferably 2 or 3, morepreferably 2.

Combinations

The peptide(s) according to the present disclosure can be used incombination with one or more additional active drugs, for instance ananti-diabetic drug, a hypolipidemic agent, an anti-obesity agent, ananti-hypertensive agent, an anti-steatotic drug, an anti-inflammatoryagent, and an agonist of peroxisome proliferator-activator receptors.

Accordingly, the present invention relates to:

-   -   a peptide or a pharmaceutical composition comprising a peptide        as disclosed herein for use in the treatment or prevention of a        disease, in combination with one or more additional active        drugs, in particular as disclosed herein; wherein the disease is        selected from the group consisting of nonalcoholic fatty liver        disease (NAFLD), non-alcoholic fatty liver (NAFL), non-alcoholic        steatohepatitis (NASH), hepatic steatosis (fatty liver), liver        inflammation, cirrhosis, hepatocellular carcinoma and fibrosis,        especially a fibrosis selected from the group consisting of a        liver fibrosis including cirrhosis, a renal fibrosis, a cardiac        fibrosis including an atrial fibrosis, an endomyocardial        fibrosis and old myocardial infarction, a pulmonary fibrosis        including cystic fibrosis and radio-induced lung fibrosis, a        vascular fibrosis such as an arterial fibrosis, a brain        fibrosis, a myelofibrosis, an arthrofibrosis, an intestinal        fibrosis, a peritoneal fibrosis, a retroperitoneal fibrosis and        a skin fibrosis;    -   a pharmaceutical composition comprising a peptide as disclosed        herein and one or more additional active drugs, in particular as        disclosed herein, for use in the treatment or prevention of a        disease selected from the group consisting of nonalcoholic fatty        liver disease (NAFLD), non-alcoholic fatty liver (NAFL),        non-alcoholic steatohepatitis (NASH), hepatic steatosis (fatty        liver), liver inflammation, cirrhosis, hepatocellular carcinoma        and fibrosis, especially a fibrosis selected from the group        consisting of a liver fibrosis including cirrhosis, a renal        fibrosis, a cardiac fibrosis including an atrial fibrosis, an        endomyocardial fibrosis and old myocardial infarction, a        pulmonary fibrosis including cystic fibrosis and radio-induced        lung fibrosis, a vascular fibrosis such as an arterial fibrosis,        a brain fibrosis, a myelofibrosis, an arthrofibrosis, an        intestinal fibrosis, a peritoneal fibrosis, a retroperitoneal        fibrosis and a skin fibrosis;    -   a product, combined preparation or kit comprising a peptide        according to the present disclosure and one or more additional        active drugs, in particular as disclosed herein, for        simultaneous, separate or sequential use in the treatment or        prevention of a disease selected from the group consisting of        nonalcoholic fatty liver disease (NAFLD), non-alcoholic fatty        liver (NAFL), non-alcoholic steatohepatitis (NASH), hepatic        steatosis (fatty liver), liver inflammation, cirrhosis,        hepatocellular carcinoma and fibrosis, especially a fibrosis        selected from the group consisting of a liver fibrosis including        cirrhosis, a renal fibrosis, a cardiac fibrosis including an        atrial fibrosis, an endomyocardial fibrosis and old myocardial        infarction, a pulmonary fibrosis including cystic fibrosis and        radio-induced lung fibrosis, a vascular fibrosis such as an        arterial fibrosis, a brain fibrosis, a myelofibrosis, an        arthrofibrosis, an intestinal fibrosis, a peritoneal fibrosis, a        retroperitoneal fibrosis and a skin fibrosis;    -   the use of a peptide for the manufacture of a medicine for the        treatment or prevention of a disease in combination with one or        more additional active drugs, in particular as disclosed herein,        wherein the disease is selected from the group consisting of        nonalcoholic fatty liver disease (NAFLD), non-alcoholic fatty        liver (NAFL), non-alcoholic steatohepatitis (NASH), hepatic        steatosis (fatty liver), liver inflammation, cirrhosis,        hepatocellular carcinoma and fibrosis, especially a fibrosis        selected from the group consisting of a liver fibrosis including        cirrhosis, a renal fibrosis, a cardiac fibrosis including an        atrial fibrosis, an endomyocardial fibrosis and old myocardial        infarction, a pulmonary fibrosis including cystic fibrosis and        radio-induced lung fibrosis, a vascular fibrosis such as an        arterial fibrosis, a brain fibrosis, a myelofibrosis, an        arthrofibrosis, an intestinal fibrosis, a peritoneal fibrosis, a        retroperitoneal fibrosis and a skin fibrosis;    -   the use of a peptide as disclosed herein and one or more        additional active drugs, in particular as disclosed herein, for        the manufacture of a medicine for the treatment or prevention of        a disease selected from the group consisting of nonalcoholic        fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL),        non-alcoholic steatohepatitis (NASH), hepatic steatosis (fatty        liver), liver inflammation, cirrhosis, hepatocellular carcinoma        and fibrosis, especially a fibrosis selected from the group        consisting of a liver fibrosis including cirrhosis, a renal        fibrosis, a cardiac fibrosis including an atrial fibrosis, an        endomyocardial fibrosis and old myocardial infarction, a        pulmonary fibrosis including cystic fibrosis and radio-induced        lung fibrosis, a vascular fibrosis such as an arterial fibrosis,        a brain fibrosis, a myelofibrosis, an arthrofibrosis, an        intestinal fibrosis, a peritoneal fibrosis, a retroperitoneal        fibrosis and a skin fibrosis;    -   a method for the treatment or prevention of a disease in a        subject, comprising administering a therapeutically effective        amount of a peptide as disclosed herein and a therapeutically        effective amount of one or more additional active drugs, in        particular as disclosed herein, wherein the disease is selected        from the group consisting of nonalcoholic fatty liver disease        (NAFLD), non-alcoholic fatty liver (NAFL), non-alcoholic        steatohepatitis (NASH), hepatic steatosis (fatty liver), liver        inflammation, cirrhosis, hepatocellular carcinoma and fibrosis,        especially a fibrosis selected from the group consisting of a        liver fibrosis including cirrhosis, a renal fibrosis, a cardiac        fibrosis including an atrial fibrosis, an endomyocardial        fibrosis and old myocardial infarction, a pulmonary fibrosis        including cystic fibrosis and radio-induced lung fibrosis, a        vascular fibrosis such as an arterial fibrosis, a brain        fibrosis, a myelofibrosis, an arthrofibrosis, an intestinal        fibrosis, a peritoneal fibrosis, a retroperitoneal fibrosis and        a skin fibrosis;    -   a method for the treatment or prevention of a disease in a        subject, comprising administering a therapeutically effective        amount of a pharmaceutical composition comprising a peptide as        disclosed herein and one or more additional active drugs, in        particular as disclosed herein, wherein the disease is selected        from the group consisting of nonalcoholic fatty liver disease        (NAFLD), non-alcoholic fatty liver (NAFL), non-alcoholic        steatohepatitis (NASH), hepatic steatosis (fatty liver), liver        inflammation, cirrhosis, hepatocellular carcinoma and fibrosis,        especially a fibrosis selected from the group consisting of a        liver fibrosis including cirrhosis, a renal fibrosis, a cardiac        fibrosis including an atrial fibrosis, an endomyocardial        fibrosis and old myocardial infarction, a pulmonary fibrosis        including cystic fibrosis and radio-induced lung fibrosis, a        vascular fibrosis such as an arterial fibrosis, a brain        fibrosis, a myelofibrosis, an arthrofibrosis, an intestinal        fibrosis, a peritoneal fibrosis, a retroperitoneal fibrosis and        a skin fibrosis.

In particular, a therapeutic or sub-therapeutic effective amount of oneor more additional active drugs can be used. By “sub-therapeutic” isintended to refer to an amount that can be for instance 90, 80, 70, 60,50, 40, 30, 20 or 10% of the conventional therapeutic dosage (inparticular for the same indication and/or the same administration routeand/or frequency of administration).

The anti-diabetic drug can be for instance insulin, insulin derivativesand mimetics; insulin secretagogues such as the sulfonylureas (e.g.,chlorpropamide, tolazamide, acetohexamide, tolbutamide, glyburide,glimepiride, glipizide); gliflozins such as empagliflozin anddapagliflozin; glyburide and Amaryl; liraglutide (NN2211);insulinotropic sulfonylurea receptor ligands such as meglitinides, e.g.nateglinide and repaglinide; thiazolidinediones (e.g., rosiglitazone(AVANDIA), troglitazone (REZULIN), pioglitazone (ACTOS), balaglitazone,rivoglitazone, netoglitazone, troglitazone, englitazone, ciglitazone,adaglitazone, darglitazone that enhance insulin action (e.g., by insulinsensitization), thus promoting glucose utilization in peripheraltissues; protein tyrosine phosphatase-IB (PTP-1B) inhibitors such asPTP-112; Cholesteryl ester transfer protein (CETP) inhibitors such astorcetrapib, GSK3 (glycogen synthase kinase-3) inhibitors such asSB-517955, SB-4195052, SB-216763, NN-57-05441 and NN-57-05445; RXRligands such as GW-0791 and AGN-194204; sodium-dependent glucosecotransporter inhibitors such as T-1095 or canagliflozin; glycogenphosphorylase A inhibitors such as BAY R3401; biguanides such asmetformin and other agents that act by promoting glucose utilization,reducing hepatic glucose production and/or diminishing intestinalglucose output; alpha-glucosidase inhibitors such as acarbose andmigiitoi) and other agents that slow down carbohydrate digestion andconsequently absorption from the gut and reduce postprandialhyperglycemia; GLP-1 (glucagon like peptide-1), GLP-1 analogs such asExendin-4 and GLP-1 mimetics; and DPPIV (dipeptidyl peptidase IV)inhibitors such as vildagliptin. It can also be an anti-diabetic drugdescribed in Expert Opin Investig Drugs 2003, 12(4): 623-633, FIGS. 1 to7 . Antidiabetic drug may also include a molecule preventing the bindingof αPKC and ALMS1 such as those disclosed in WO 2015/114062, thedisclosure thereof being incorporated herein by reference.

The hypolipidemic agent can be for instance 3-hydroxy-3-methyl-glutarylcoenzyme A (HMG-CoA) reductase inhibitors, e.g. lovastatin,pitavastatin, simvastatin, pravastatin, cerivastatin, mevastatin,velostatin, fluvastatin, dalvastatin, atorvastatin, rosuvastatin andrivastatin; squalene synthase inhibitors; FXR (farnesoid X receptor) andLXR (liver X receptor) ligands such as obeticholic acid; bile acidsequenstrants, such as cholestyramine and colesevelam; fibrates;nicotinic acid and aspirin; aramchol, a transmembrane G protein-coupledreceptor (TGR) 5 agonist.

The anti-obesity agent can be for instance orlistat, rimonabant,phentermine, topiramate, qnexa, and locaserin.

The anti-hypertensive agent can be for instance loop diuretics such asethacrynic acid, furosemide and torsemide; angiotensin converting enzyme(ACE) inhibitors such as benazepril, captopril, enalapril, fosinopril,lisinopril, moexipril, perinodopril, quinapril, ramipril andtrandolapril; inhibitors of the Na—K-ATPase membrane pump such asdigoxin; neutralendopeptidase (NEP) inhibitors such as sacubitril;ACE/NEP inhibitors such as omapatrilat, sampatrilat and fasidotril;angiotensin II antagonists such as candesartan, eprosartan, irbesartan,losartan, telmisartan and valsartan, in particular valsartan;combinations of NEP inhibitors and angiotensin II antagonists such assacubitril and valsartan (i.e. Entresto); renin inhibitors such asditekiren, zankiren, terlakiren, aliskiren, RO 66-1132 and RO-66-1168;beta-adrenergic receptor blockers such as acebutolol, atenolol,betaxolol, bisoprolol, metoprolol, nadolol, propranolol, sotalol andtimolol; inotropic agents such as digoxin, dobutamine and milrinone;calcium channel blockers such as amlodipine, bepridil, diltiazem,felodipine, nicardipine, nimodipine, nifedipine, nisoldipine andverapamil; aldosterone receptor antagonists; and aldosterone synthaseinhibitors.

The agonist of peroxisome proliferator-activator receptors can be forinstance fenofibrate, pioglitazone, rosiglitazone, tesaglitazar,BMS-298585, L-796449, the compounds specifically described in the patentapplication WO 2004/103995 i.e. compounds of examples 1 to 35 orcompounds specifically listed in claim 21, or the compounds specificallydescribed in the patent application WO 03/043985 i.e. compounds ofexamples 1 to 7 or compounds specifically listed in claim 19 andespecially(R)-1-{4-[5-methyl-2-(4-trifluoromethyl-phenyl)-oxazol-4-ylmethoxy]-benzenesulfonyl}-2,3-dihydro-1H-indole-2-carboxylicor a salt thereof.

Other drugs of interest can be for instance cenicriviroc, simtuzumab,selonsertib, emricasan.

In a particular embodiment, the one or more additional active drugs usedin combination with the peptide can be selected among: a GLP-1 analogsuch as liraglutide, obeticholic acid, a gliflozin, simtuzumab (GS6624), cenicriviroc, aramchol, a Galectin 3 inhibitor such as GR-MD-02,a TGR5 agonist and a dual FXR/TGR5 agonist such as INT-777 or INT-767,and emricasan.

The anti-inflammatory agent can be any drug known by the skilled personsuch as nonsteroidal anti-inflammatory agents (NSAIDs), includingsalicylic acid, ibuprofen in its various forms and naproxen in itsvarious forms, a steroidal anti-inflammatory such as corticosteroids, ananti-inflammatory anti-TNF alpha antibody and combinations thereof.

The form of the pharmaceutical compositions, the route ofadministration, the dosage and the regimen naturally depend upon thecondition to be treated, the severity of the illness, the age, weight,and sex of the patient, etc.

The pharmaceutical or therapeutic compositions of the present disclosurecan be formulated for a topical, oral, parenteral, intranasal,intravenous, intramuscular, subcutaneous or intraocular administrationand the like.

The peptide used in the pharmaceutical composition of the presentdisclosure is present in a therapeutically effective amount.

The pharmaceutical composition comprising the peptide is formulated inaccordance with standard pharmaceutical practice (Lippincott Williams &Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J.Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York) known bya person skilled in the art.

In one aspect, the present invention provides a stable formulation forparenteral injection of the pharmaceutical composition according to thepresent disclosure comprising a peptide or a salt thereof, wherein thepeptide has been dried and then is reconstituted in a solvent prior touse. The peptide (or, in embodiments where the formulation comprises twoor more peptides, each of the peptides) is mixed with a non-volatilebuffer and dried to a dry peptide powder. Suitable buffers include, butare not limited to, glycine buffers, citrate buffers, phosphate buffers,and mixtures thereof. In one embodiment, the buffer is a glycine buffer.In another embodiment, the buffer is a mixture of citrate buffer andphosphate buffer. In some embodiments, wherein the formulation comprisestwo or more peptides, the first and second buffer are the same. In someembodiments, wherein the formulation comprises two or more peptides, thefirst and the second buffer are different. Alternatively, thepharmaceutical composition according to the present disclosure may bestored in an aqueous state. The solution may contain, if desired,further additives or excipients, which must be compatible with theactive principle and, if they are not removed during the freeze-dryingstage, they must also be compatible with the route of administration.For parenteral administration, the composition may be injectedintradermally, subcutaneously, intramuscularly, or intravenously.Preferably, the composition or peptide is injected or to be injectedsubcutaneously, in particular in the fat tissue.

It may preferably be placed with a mini-osmotic pump or other controlleddelivery device implanted into the body. Preferably, it may be mixedwith other compounds to make a depot slow release formulation. Apreferred route of administration is subcutaneous injection, forinstance by using a disposable or multiunit dispensing device, similarto an insulin pen. The peptide can also be administered by a deviceallowing the subcutaneous administration without any needle, anon-invasive system.

In addition, the peptide can be administered by using any drug deliverysystem available. In particular, the use of recombinant humanhyaluronidase enzyme, rHuPH20, to enable and optimize subcutaneous drugdelivery for appropriate co-administered therapies is contemplated.

With the technology, some biologics and compounds that are administeredintravenously may instead be delivered subcutaneously, or under theskin, potentially providing a better experience for patients, andincreasing health system efficiency by reducing administration time,injection pain and infusion site reactions.

In one embodiment, the peptide of the present disclosure may be mixedwith other compounds to make a depot slow release formulation. This maythen be injected subcutaneously to form a slow release depot.

For oral administration, the composition can be formulated intoconventional oral dosage forms such as tablets, capsules, powders,granules and liquid preparations such as syrups, elixirs, andconcentrated drops. Non-toxic solid carriers or diluents may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharine, talcum, cellulose,glucose, sucrose, magnesium, carbonate, and the like. For compressedtablets, binders, which are agents which impart cohesive qualities topowdered materials, are also necessary. For example, starch, gelatine,sugars such as lactose or dextrose, and natural or synthetic gums can beused as binders. Disintegrants are also necessary in the tablets tofacilitate break-up of the tablet. Disintegrants include starches,clays, celluloses, algins, gums and crosslinked polymers. Moreover,lubricants and glidants are also included in the tablets to preventadhesion to the tablet material to surfaces in the manufacturing processand to improve the flow characteristics of the powder material duringmanufacture. Colloidal silicon dioxide is most commonly used as aglidant and compounds such as talc or stearic acids are most commonlyused as lubricants.

For transdermal administration, the composition can be formulated intoointment, cream or gel form and appropriate penetrants or detergentscould be used to facilitate permeation, such as dimethyl sulfoxide,dimethyl acetamide and dimethylformamide.

For transmucosal administration, nasal sprays, intrapulmonaryinhalation, rectal or vaginal suppositories can be used. In oneembodiment, the invention may be administered by the intrapulmonaryroute using either a dry powder or liquid formulation administered usingan intrapulmonary drug delivery device according to methods known in theart. The active compound can be incorporated into any of the knownsuppository bases by methods known in the art. Examples of such basesinclude cocoa butter, polyethylene glycols (carbowaxes), polyethylenesorbitan monostearate, and mixtures of these with other compatiblematerials to modify the melting point or dissolution rate.

Pharmaceutical compositions according to the present disclosure may beformulated to release the active drug substantially immediately uponadministration or at any predetermined time or time period afteradministration.

Pharmaceutical compositions according to the present disclosure cancomprise one or more peptides of the present disclosure associated withpharmaceutically acceptable excipients and/or carriers. These excipientsand/or carriers are chosen according to the form of administration asdescribed above.

In a particular embodiment, the pharmaceutical composition according tothe present disclosure comprises between 0.01 ng and 10 g of the peptideof the present disclosure. In one embodiment, pharmaceutical compositionaccording to the present disclosure comprises between 0.1 ng and 1 g ofthe peptide of the present disclosure.

All the references cited in this application, including scientificarticles and summaries, published patent applications, granted patentsor any other reference, are entirely incorporated herein by reference,which includes all the results, tables, figures and texts of thesesreferences.

Although having different meanings, the terms “comprising”, “having”,“consisting in” and “containing” can be replaced one for the other inthe entire application. Further aspects and advantages of the presentdisclosure will be described in the following examples, which should beregarded as illustrative and not limiting.

EXAMPLES Example 1: Effect of Adipose Tissue Targeted PATAD Treatment onExpression Levels of Key Fatty Acids Transporters and Receptors 11 DaysPost Injection

As PATAD subcutaneous injection relates to restoration of glucoseabsorption in the adipose tissue, the inventors measured the expressionlevels of all 6 isoforms for the FATPs (1-6) (Fatty acid transportprotein 1-6) and the 4 isoforms of the FFAR (Free Fatty Acid Receptor)to assess any effect on these genes.

Interestingly, PATAD injections induced a very specific and drasticdecrease in FAPT2 in the adipose tissue allowing to correlate the effectof PATAD treatment directly on the FATP2 expression drop (FIG. 1A).Since it was previously shown that PATAD peptide does not circulate inthe body, that its effect was limited to the adipose tissue and alsobased on its mechanism of action which is to interfere with ALMS1-PKCinteraction, PATAD's novel action in the adipose tissue is to decreaseFATP2. The expression levels of the transporters and receptors in thetwo other major organs can be related to indirect effect (FIGS. 1B-C).

Example 2: ADPIF Peptide is More Active than PATAD Peptide in DecreasingFATP2 Expression Levels in the Adipose Tissue

Mice were injected with a single dose of either scramble peptide orPATAD or ADPIF. 11 days post injection, the mice were euthanized andadipose tissue was sampled for RNA extraction followed by real-time PCR.The expression level of FATP2 is similar to the lean control whereasPATAD, although effective in reducing FATP2 expression levels in theadipose tissue, is less effective than ADPIF (FIG. 2A).

Example 3: ADPIF is Effective in Increasing Circulating Levels of GLP-111 Days Post Adipose Tissue Injection

Mice were injected with a single dose of either scramble peptide orPATAD or ADPIF. 11 days post injection, the mice were euthanized, andplasma was obtained and used for determination of circulating GLP1 frommice with the indicated treatment. Both PATAD and ADPIF restored highcirculating levels of GLP1, with ADPIF inducing a greater increase thanPATAD (FIG. 2B).

Example 4: Protective Effect on Liver after 3 Months of a SingleInjection of Adipose Tissue Targeted Either PATAD or ADPIF Treatment

Mice presented were euthanized and their plasma and organs were sampledfor analysis. The ratio of liver weight to body weight versus age wasdetermined and presented in FIG. 3B. The size of the liver in responseto the PATAD treatment was clearly decreased compared to controls thatwere DIO diabetic male controls that received only the vehicle.Cryosection of the livers were then stained with Adipored and DAPI todetect the level of lipid droplets. Mice treated with PATAD or ADPIFclearly showed a decrease in the size of the hepatic lipid droplets(FIG. 3A, right panel; FIG. 7 , right panel). Hepatic function isassessed by two liver related strong biomarkers namely (AST (AspartateAminotransferase) and ALT (Alanine Aminotransferase)), whose levelsincrease proportionately with liver damage. Interestingly, mice treatedwith PATAD or ADPIF (after a 3-month period with a single subcutaneouspeptide injection) exhibit a dramatic decrease in these two robusthepatic biomarkers (FIG. 3C) indicating a protective effect of PATAD andADPIF on the liver related to a decrease in the phenomenon of steatosis.PATAD and ADPIF are both effective in reducing circulating levels of ASTand ALT. ADPIF is more active in reducing circulating levels of ALT,which translates improvement of liver cell injury.

Example 5: ADPIF Peptide is More Active than PATAD Peptide in PreventingHyperglycemia

These are results obtained from series of glucose tolerance test (GTT)and data from two-time points namely 30 minutes and 120 minutes wereused to generate these curves for both control and PATAD treated dietinduced obese (DIO) and diabetic wild type male C57/B6 mice (control).

These data show that the peptide-based ADPIF have a better effect inglucose tolerance test than the PATAD peptide (FIG. 4 ).

Example 6. ADPIF Peptide is More Active than PATAD Peptides in ReducingHepatic Levels of LOXL2 and FABP4 Proteins

LOXL2 is the enzyme promoting the development of fibrotic lesions byfavoring the polymerization of collagen and FABP4 is a protein involvedin lipid metabolism whose level increases with increasing lipiddeposition in the liver. 3 months post treatment with a uniqueinjection, liver extracts from the mice with the indicated mice wereused and these data show that peptide ADPIF is more potent than PATAD indecreasing the hepatic levels of LOXL2, thereby decreasing the abilityto generate more fibrotic lesions in the liver and in decreasing thelevels of FABP4, which correlates with a decrease in liver triglycerideslevels (FIG. 5 ).

Example 7: Circulating Lipid Profile in Response to ADPIF Treatment inthe Adipose Tissue

ADPIF adipose tissue injection has proven to be unexpectedly liverprotective relative to a specific decrease in expression levels of FATP2in the adipose tissue and an associated profile of the FATPs and FFARsin the muscles and liver. As these changes impact circulating populationof the different lipids. Ceramides are a group of biological activelipids known to be involved in NAFLD. We measured and determined theeffect of ADPIF injections on the ceramides profile following 3injections of ADPIF at a frequency of one injection per week in thesubcutaneous adipose tissue. ADPIF globally induces a decrease in thehepatic ceramides content with variations between the differentceramides (FIG. 6 ).

Example 8: ADPIF is Effective in Decreasing the Size of the LipidDroplets in the Liver

In FIG. 7 , cryosections from livers of treated mice (with same dosageregiment as in example 6) were stained for lipid droplets whichevidenced a global decrease in the sizes of the lipid droplets in theliver post ADPIF treatment thereby indicating a decrease in theaccumulation of lipids in the liver.

Example 9: ADPIF Treatment in the Adipose Tissue Surprisingly Repressedthe Expression Levels of Key Lipogenesis and Modulating Enzymes in theLiver

ACC and FASN form part of the lipogenesis pathway by which fatty acidsare synthesized in the liver, together with the Srebf1 which is a keyprotein involved in lipid handling. Following ADPIF treatment, theseenzymes were repressed indicating that ADPIF action following itsadministration in the adipose tissue is able to block de novolipogenesis in the liver (FIG. 8 ).

Examples 10: ADPIF Treatment in the Adipose Tissue Blocks theProgression of Hepatic Fibrosis in DIO-NASH Mouse Model

Liver cryosections from ADPIF treated mice (same as in example 8) wereused for immunodetection of either LOXL2 or Collagen IV and showed thatADPIF was effective in preventing the progression of fibrotic markers(FIG. 9A). The levels of secreted LOXL2 in the plasma was alsosignificantly reduced (FIG. 9B). As a results of the decrease in LOXL2and the decrease of collagen depots, fibrotic lesions readily detectedin the untreated DIO-NASH liver (FIG. 9C, left panel) on transmittedelectron microscopy were no longer detected in ADPIF treated livers.

Example 11. ADPIF Selectively Reduces Targeted Lysophosphatidylcholines(LPC) in the Liver and Other Tissues

Lipid contents for LPCs were analyzed 3 months post ADPIF injection inthe adipose tissue to see how ADPIF could impact the levels of LPCs,known substrate for autotaxin, a key secreted enzyme playing a role infibrosis progression. Surprisingly, ADPIF was effective in reducingselective LPCS, in the tested organs with LPC18:2 presenting the mostprominent reduction in the pancreas, adipose tissue and liver (FIG. 10).

Example 12. ADPIF Peptide is Effective in Preventing Collagen IV Depotsin the Kidney Hence Protecting the Kidney from Fibrosis

Cryosections of kidneys from DIO vehicle treated and DIO ADPIF treatedmice 3 months post treatment, were immunostained for collage IVdeposition and tight junction with a ZO-1 antibody to assess the effectof ADPIF peptide on fibrosis progression in other soft tissues.

Surprisingly, a clear reduction of Collagen IV was observed in thekidneys of the ADPIF-treated mice compared to the vehicle treated miceindicating that ADPIF peptide was also effective in protecting thekidney from fibrosis (FIG. 11 ).

Materials & Methods Mice Husbandry

For this study, mice were on a C57/BL6 genetic background. All animalswere housed in a temperature and humidity controlled facility, with a 12h-light/12 h-dark cycle fed during the whole phase mice will be fed witha 60% high fat diet from Research Diets (D12492), and tap water will beprovided ad libitum. Mice were fed and tested regularly for glucosetolerance test for their glucose tolerance and once they were glucoseintolerant they were used for treatment.

Peptide Sequences and Synthesis

PATAD is the name given to a series of peptides derived from the PKCalpha isoform that are biologically active with the ability to triggerglucose absorption specifically in the adipose tissue.

Stapled peptide sequence A: VE CTM-[2-(4-pentenyl) alanine]-EK RVLA-[2-(4-pentenyl) alanine]-L DKP PFL TQL HS (SEQ ID NO: 49)

-   -   Stapled peptide sequence B: S-[2-(4-pentenyl) alanine]-CKG        LMT-[2-(4-pentenyl) alanine]-HP AKR LGC GPE G (SEQ ID NO: 50)    -   Scrambled peptide sequence A:

(SEQ ID NO: 51) KEVPVDTCHLTLMLLFRSVALKQHPE

-   -   Scrambled peptide sequence B: SAECKGRHGTPPGKLMICKGL (SEQ ID NO:        22)    -   ADPIF is the name given to a series of peptides derived from the        PKC alpha isoform derivate to PATAD peptide and which present        two specific mutations.    -   Stapled peptide ADPIF sequence:

(SEQ ID NO: 32) VECTT R EKEVLA S LDKAAFLTQLHS

-   -   wherein R and S carry the stapling, being preferably        2-(7-octenyl)arginine and 2-(4-pentenyl)serine, respectively.

The stapled and scrambled peptides were purchased from CPC, USA with a95% purity.

All peptides were initially dissolved in DMSO and then diluted insterile saline solution at a concentration of 10 ng/μL. 2.5 μL of eachpeptide (stapled or scrambled) were mixed and then injected directly inthe subcutaneous adipose tissue in each mouse.

Dosage Regimen of the Peptides

Control mice were injected either with the Scramble controls or withvehicle (0.9% saline solution) (retroperitoneal fat/subcutaneousinjection: one injection at DO and the indicated tests performed.Treated mice were injected with the mixture of two PATAD stapledpeptides and with ADPIF stapled peptide following the same procedurewith the PATAD peptides and with ADPIF peptide test item(retroperitoneal fat/subcutaneous injection: one injection at DO and theindicated tests performed). In a second series of experiment, ADPIF wasinjected once per week at a dosage of 25 ug per mouse in thesubcutaneous adipose tissue in the retroperitoneal area for 3 weeks andthe mice were euthanized one week after the last injection. Mice wereeuthanized and tissue samples and plasma were isolated and kept at −80°C. for further testings.

RNA Extraction, cDNA Synthesis, q-PCR and Taqman

Total RNA was prepared from the different tissues and cells using aRiboPure™ kit (Catalog #: AM1924; Ambion) followed by a DNAse treatmentwith the TURBO DNA-free™ (Catalog #: AM 1907; Ambion). RNA integrity wasassessed by gel electrophoresis and RNA concentration by EppendorfBiophotometer Plus with the Hellma® Tray Cell (Catalog #: 105.810-uvs;Hellma). Reverse transcription of 1 μg total RNA to cDNA was performedusing the BioRadiScript™ cDNA synthesis kit (Catalog #: 170-8891;BioRad). Real-time quantitative polymerase chain reaction amplificationwas performed in a BioRad CFX96™ Real-Time System using the iQ™ SYBR®Green Supermix (Catalog #: 170-8886; BioRAd) and primer sets optimizedfor tested targets for SYBR Green-based real-time PCR for the real-timePCR. Taqman analysis was carried out with the specific gene assay withthe Taqman® Fast Advanced Master Mix (Catalog #: 4444557; AppliedBiosystems). The normalized fold expression of the target gene wascalculated using the comparative cycle threshold (C_(t)) method bynormalizing target mRNA C_(t) to those for GAPDH using the CFX ManagerSoftware Version 1.5 and was verified using the Lin-Reg program (Ruijteret al., 2009). All primer pairs were purchased from either Biorad orfrom Quantitect.

Primer specifications are found in the table below:

SEQ Gene ID Fragment name Primer name Sequences NO: size Fatp1Mu_Slc27a1- TGCTTTGGTT 37 156 bp RT-ex3F TCTGGGACTT Mu_Slc27a1-GCTCTAGCCG 38 RT-ex4R AACACGAATC Fatp2 Mu_Slc27a2- TGGACAAAGT 39 165 bpRT-ex4F AGACGGAGTG TC Mu_Slc27a2- TAGCAAGGCC 40 RT-ex5R TGTCCCATAC Fatp3Mu_Slc27a3- TGAGAACTTG 41 171 bp RT-ex9F CCACCGTATG Mu_Slc27a3-GGCAGGTAGG 42 RT-ex10R CCCCTATATC Fatp4 Mu_Slc27a4- GTTTCATCCG 43 184 bpRT-ex2F GGTCTTCATC Mu_Slc27a4- GTGTCTGTGC 44 RT-ex3R CCTCGAAAAT Fatp5Mu_Slc27a5- AAGTTCTCTG 45 191 bp RT-ex4F CCTCCCGATT Mu_Slc27a5-CAAAGCGTTG 46 RT-ex5R CTGGAAGTTT Fatp6 Mu_Slc27a6- TCGATTCCCT 47 204 bpRT-ex1F CCTACACTGC Mu_Slc27a6- TTGGTGGTAC 48 RT-ex2R TGGCTCATCAPrimers from Quantitect were: Srebf1: QT00167055, Acc: QT01554441 andfor Fasn: QT00149240.

AdipoRed Staining of Liver Sections

Livers were isolated and briefly washed in PBS buffer (pH 7.4). afterweighing the dried liver, for the liver to body weight ratio, a slicedsample of the liver were then placed in 4% paraformaldehyde (in 0.1Msodium phosphate buffer, pH 7.2) for 15 min, washed in PBS and incubatedin AdipoRed dye (1/25; Lonza, Switzerland) with 30 μM DAPI(Sigma-Aldrich, USA) for 15 minutes. After 3 washes with PBS sampleswere mounted on slides and pictures were taken using Zeiss microscope.

Immunofluorescence Experiments

For immunofluorescence experiments, liver and kidneys freshly sampledwere included in Optimal Cutting Temperature Compound™ (OCT™, Catalog#4583, Tissue-Tek® OCT™, Sakura® Finetek, Torrance, California, USA) andcryosections of 7 μm were cut with Cryostat Leica CM1950. Cryosectionswere washed with 1×PBS and fixated in 4% formaldehyde solution for 15min (Catalog #: F555-4L, Sigma-Aldrich, Saint-Louis, Missouri, USA) andthen permeabilized with 0.02% SDS-PBS for 30 seconds. Blocking solutionwas 5%-Bovine Serum Albumin (BSA) in PBS. Primary antibodies werediluted in blocking solution and incubated overnight and indicatedsecondary antibodies were diluted in PBS for 30 minutes. Nuclei werecounterstained with Hoechst (Catalog #: D1306, Invitrogen, Carlsbad,California, USA). Slides were then mounted with Vectashield® MountingMedium (Catalog #: H-1000, Vector Laboratories, Burlingame, California,USA). Images were acquired and analyzed with Zeiss Imager.Z2 microscopeequipped with either Zeiss AxioVision or Zeiss ZEN 2012 software (CarlZeiss Inc., Oberkochen, Germany).

Anti-Collagen IV antibody, ab6586 from Abcam and LOXL2 antibody,GTX105085 from GeneTex were used.

For the electron microscopy imaging on the liver extracts, the sampleswere immersed in glutaraldehyde (2.5%) and paraformaldehyde (2.5%) incacodylate buffer (0.1 M, pH 7.4). The samples were post-fixed in 1%osmium tetroxide, dehydrated through graded alcohol (50, 70, 90, and100%) and propylene oxide for 30 minutes each, and embedded in Epon 812.Semithin sections were cut at 2 μm on an ultra microtome (Leica UltracutUCT) and ultrathin sections were cut at 70 nm and contrasted with uranylacetate and lead citrate and examined at 70 kv with a Morgagni 268Delectron microscope. Images were captured digitally by Mega View IIIcamera (Soft Imaging System).

Biochemical Assay For the Plasma Determination AST and ALT ELISAMeasurements

Plasma samples from the indicated mice were used to determine plasmacontent of either AST (aspartate aminotransferase) or ASL (alanineaminotransferase) both robust indicators of liver damage were measuredusing commercially purchased ELISA kits. These parameters weredetermined according to manufacturer's procedure.

-   -   SEB214Mu (96 Tests): Enzyme-linked Immunosorbent Assay Kit For        Aspartate Aminotransferase (AST), Cloud Clone Corp.    -   SEA207Mu (96 Tests): Enzyme-linked Immunosorbent Assay Kit For        Alanine Aminotransferase (ALT), Cloud Clone Corp.

For LOXL2 and GLP-1 measurements, the two commercially available kits:Lysyl Oxydase Like Protein 2 (LOXL2), Enzyme-linked Immunosorbent AssayKit, Mus musculus, SEF552Mu, Cloud Clone Corp;

RayBio Human/Mouse/Rat GLP-1 Enzyme Immunoassay Kit, EIA-GLP1,RayBiotech were used with the indicated procedures from themanufacturer.

For the measurements on the liver extracts, the same kit for LOXL2 wasused and for FABP4, the commercial kit used was the Elisa kit for FattyAcid Binding Protein 4, Adipocyte (FABP4), SEB693Mu, Cloud Clone Corp.

For the lipid composition in the different tissues and plasma, allsamples were flashed frozen at the time of sampling, immediately aftersacrifice, and were sent to the lipidomic core facility of Dijon,(Plateforme de Lipidomique-uBorgogne INSERM UMR866/Labex LipSTIC).

Example 13. In Vivo Efficacy Study of ADPIF Peptide in STAM Model ofNon-Alcoholic Steatohepatitis Materials & Methods Test Substances

The peptide was ADPIF peptide as described above. The dosing solutionwas prepared according to the formulation instructions.

Induction of NASH

NASH was induced in 12 male mice by a single subcutaneous injection of200 μg streptozotocin (STZ, Sigma-Aldrich, USA) solution 2 days afterbirth and feeding with high fat diet (HFD, 57 kcal % fat, Cat #HFD32,CLEA Japan, Inc., Japan) after 4 weeks of age.

Route of Drug Administration

The peptide was administered subcutaneously in the adipose tissue in avolume of 100 mL per mouse.

Treatment Dose

The peptide was administered at a dose of 25 microgram (μg) per mouseonce weekly.

Animals

C57BL/6 mice (14-day-pregnant female) were obtained from Japan SLC, Inc.(Japan). All animals used in the study were housed and cared for inaccordance with the Japanese Pharmacological Society Guidelines forAnimal Use.

The animals were maintained in a SPF facility under controlledconditions of temperature (23±2° C.), humidity (45±10%), lighting(12-hour artificial light and dark cycles; light from 8:00 to 20:00) andair exchange. A high pressure was maintained in the experimental room toprevent contamination of the facility.

The animals were housed in TPX cages (CLEA Japan) with a maximum of 3mice per cage. Sterilized Paper-Clean (Japan SLC) was used for beddingand replaced once a week.

Sterilized solid HFD was provided ad libitum, being placed in a metallid on the top of the cage. Pure water was provided ad libitum from awater bottle equipped with a rubber stopper and a sipper tube. Waterbottles were replaced once a week, cleaned, and sterilized in anautoclave and reused.

Mice were identified by ear punch. Each cage was labeled with a specificidentification code.

Measurement of Plasma Biochemistry

For plasma biochemistry, non-fasting blood was collected inpolypropylene tubes with anticoagulant (Novo-Heparin, MochidaPharmaceutical Co. Ltd., Japan) and centrifuged at 1,000×g for 15minutes at 4° C. The supernatant was collected and stored at −80° C.until use. Plasma ALT level was measured by FUJI DRI-CHEM 7000(Fujifilm, Japan).

Measurement of Liver Triglyceride Content

Liver total lipid-extracts were obtained by Folch's method (Folch J. etal., J. Biol. Chem. 1957; 226: 497). Liver samples were homogenized inchloroform-methanol (2:1, v/v) and incubated overnight at roomtemperature. After washing with chloroform-methanol-water (8:4:3,v/v/v), the extracts were evaporated to dryness, and dissolved inisopropanol. Liver triglyceride content was measured by TriglycerideE-test (Wako Pure Chemical Industries, Ltd., Japan).

Histological Analyses

For HE staining, sections were cut from paraffin blocks of liver tissueprefixed in Bouin's solution and stained with Lillie-Mayer's Hematoxylin(Muto Pure Chemicals Co., Ltd., Japan) and eosin solution (Wako PureChemical Industries). NAFLD Activity score (NAS) was calculatedaccording to the criteria of Kleiner (Kleiner DE. et al., Hepatology,2005; 41:1313).

To visualize collagen deposition, Bouin's fixed liver sections werestained using picro-Sirius red solution (Waldeck, Germany). Forquantitative analysis of fibrosis area, bright field images of Siriusred-stained sections were captured around the central vein using adigital camera (DFC295; Leica, Germany) at 200-fold magnification, andthe positive areas in 5 fields/section were measured using ImageJsoftware (National Institute of Health, USA).

Sample Collection

For plasma samples, the remaining plasma was collected and stored at−80° C. for further analysis.

For liver samples, left lateral lobe was collected and cut into 6pieces. Two pieces of left lateral lobe, left and right medial lobes,and caudate lobe were snap frozen in liquid nitrogen and stored at −80°C. for further analysis. The other 2 pieces of left lateral lobe werefixed in Bouin's solution and then embedded in paraffin. Samples werestored at room temperature for histology. The remaining pieces of leftlateral lobe were embedded in O.C.T. compound and quick frozen in liquidnitrogen. Samples were stored at −80° C. for further analysis.

Statistical Tests

Statistical analyses were performed using Student's t-test on GraphPadPrism 6 (GraphPad Software Inc., USA). P values<0.05 were consideredstatistically significant. A trend or tendency was assumed when aone-tailed t-test returned P values<0.1. Results were expressed asmean±SD.

Experimental Design and Treatment Study Groups Group 1: ADPIF Peptide

Six NASH mice were subcutaneously in the adipose tissue administeredvehicle supplemented with ADPIF peptide at a dose of 25 mg per mouseonce weekly from 4 to 9 weeks of age.

Group 2: Vehicle

Six NASH mice were subcutaneously in the adipose tissue administeredvehicle [DMSO in saline] in a volume of 100 mL per mouse once weeklyfrom 4 to 9 weeks of age. The table below summarizes the treatmentschedule:

Test Dose Volume Sacri- No. sub- (μg per (μl per Regi- fice Group miceMice stance mouse) mouse) men (wks) 1 6 STAM Test 25 100 SC, QW, 9peptide 4-9 wks 2 6 STAM Vehicle — 100 SC, QW, 9 4-9 wks

Animal Monitoring and Sacrifice

The viability, clinical signs and behavior were monitored daily. Bodyweight was recorded before the treatment. Mice were observed forsignificant clinical signs of toxicity, moribundity and mortalityapproximately 60 minutes after each administration. The animals weresacrificed at 9 weeks of age by exsanguination through direct cardiacpuncture under isoflurane anesthesia (Pfizer Inc.).

Results

Body weight changes and general condition

-   -   Body weight changes

Mean body weight in all groups gradually increased during the treatmentperiod. There were no significant differences in mean body weigh at anyday during the treatment period between the ADPIF peptide group and theVehicle group.

There were no dead animals in all groups during the treatment period. Inthe present study, none of the animals showed deterioration in generalcondition.

-   -   Liver triglyceride

The ADPIF peptide group showed a significant decrease in livertriglyceride content compared with the Vehicle group.

TABLE 1 Biochemistry Parameter Test peptide Vehicle (mean ± SD) (n = 6)(n = 6) Plasma ALT (U/L) 54 ± 9  55 ± 7  Liver triglyceride (mg/g liver)35.1 ± 17.8 75.5 ± 33.0

-   -   Sirius red staining and the fibrosis area

Liver sections from the Vehicle group showed increased collagendeposition in the pericentral region of liver lobule. The ADPIF peptidegroup showed a significant decrease in the fibrosis area (Siriusred-positive area) compared with the Vehicle group.

TABLE 2 Fibrosis area Parameter Test peptide Vehicle (mean ± SD) (n = 6)(n = 6) Sirius red-positive area (%) 0.62 ± 0.17 0.92 ± 0.23

CONCLUSION

Treatment with ADPIF peptide showed significant decrease in livertriglyceride content and the fibrosis area compared with the Vehiclegroup. ADPIF peptide significantly reduced the fibrosis area comparedwith the Vehicle group, demonstrating an anti-fibrosis effect in thepresent study.

In conclusion, ADPIF peptide showed anti-fibrosis effects in this NASHmodel.

1-19. (canceled)
 20. A peptide wherein the peptide sequence comprises:the peptide is capable of decreasing the FATP2 expression in adiposetissue; the peptide does not simultaneously comprise one methionine, oneproline and one arginine; the peptide adopts a secondary structure whichis a helix; the peptide has a length of less than 60 amino acids; thepeptide sequence comprises a sequence selected from the group consistingof VECTMVEKRVLA (SEQ ID NO: 3); VECTXVEKRVLA (SEQ ID NO: 9);VECTMVEKXVLA (SEQ ID NO: 10); and VECTXVEKXVLA (SEQ ID NO: 11); thesequence optionally comprising from 1 to 3 modification(s) of an aminoacid selected from substitution(s), deletion(s), addition(s), and amixture thereof; wherein X is any amino acid except M, P and R; and thepeptide is modified by a chemical cross-linking.
 21. The peptide ofclaim 20, wherein the peptide has a length of less than 40 amino acids,or less than 30 amino acids, or less than 25 amino acids.
 22. Thepeptide of claim 20, wherein the peptide sequence comprises a sequenceselected from the group consisting of VECTMVEKRVLA (SEQ ID NO: 3);VECTXVEKRVLA (SEQ ID NO: 9); VECTMVEKXVLA (SEQ ID NO: 10); andVECTXVEKXVLA (SEQ ID NO: 11); wherein X is any amino acid except M, Pand R.
 23. The peptide of claim 20, wherein the peptide sequencecomprises a sequence selected from the group consisting of: a)VECTXVEKXVLALLDKXXFLTQLHS (SEQ ID NO: 20) wherein X is any amino acidexcept M, P and R, optionally comprising from 1 to 5 or from 1 to 3modification(s) of an amino acid selected from substitution(s),deletion(s), addition(s), and a mixture thereof; b)VECTMVEKRVLALLDKXXFLTQLHS (SEQ ID NO: 21) wherein X is any amino acidexcept M, P and R, optionally comprising from 1 to 5 or from 1 to 3modification(s) of an amino acid selected from substitution(s),deletion(s), addition(s), and a mixture thereof; c)VECTXVEKRVLALLDKPPFLTQLHS (SEQ ID NO: 22) wherein X is any amino acidexcept M, P and R, optionally comprising from 1 to 5 or from 1 to 3modification(s) of an amino acid selected from substitution(s),deletion(s), addition(s), and a mixture thereof; d)VECTMVEKXVLALLDKPPFLTQLHS (SEQ ID NO: 23) wherein X is any amino acidexcept M, P and R, optionally comprising from 1 to 5 or from 1 to 3modification(s) of an amino acid selected from substitution(s),deletion(s), addition(s), and a mixture thereof; and e) a peptidecomprising a sequence of any segment of at least 5 to 25 consecutiveresidues of any sequence a) to d).
 24. The peptide of claim 20, whereinthe peptide sequence comprises a sequence selected from the groupconsisting of: (SEQ ID NO: 24) VECTM X EKRVLA X ; (SEQ ID NO: 25) VECTXX EKRVLA X ; (SEQ ID NO: 26) VECTM X EKXVLA X ; (SEQ ID NO: 27) VECTX XEKXVLA X ; (SEQ ID NO: 28) VECTX X EKXVLA X LDKXXFLTQLHS;(SEQ ID NO: 29) VECTM X EKRVLA X LDKXXFLTQLHS; (SEQ ID NO: 30) VECTX XEKRVLA X LDKPPFLTQLHS; (SEQ ID NO: 31) VECTM X EKXVLA X LDKPPFLTQLHS;

wherein the residues which are bold and underlined X carry the staplingand is any amino acid derivative suitable for stapling; and wherein X isany amino acid except M, P and R; with the sequence having optionallyfrom 1 to 5 or from 1 to 3 modification(s) of an amino acid selectedfrom substitution(s), deletion(s), addition(s), and a mixture thereof.25. The peptide of claim 20, wherein the peptide sequence comprises asequence selected from the group consisting of: a)VECTXVEKXVLALLDKXXFLTQLHS (SEQ ID NO: 20) wherein X is any amino acidexcept M, P and R; b) VECTMVEKRVLALLDKXXFLTQLHS (SEQ ID NO: 21) whereinX is any amino acid except M, P and R; c) VECTXVEKRVLALLDKPPFLTQLHS (SEQID NO: 22) wherein X is any amino acid except M, P and R; d)VECTMVEKXVLALLDKPPFLTQLHS (SEQ ID NO: 23) wherein X is any amino acidexcept M, P and R; and e) a peptide comprising a sequence of any segmentof at least 5 to 25 consecutive residues of any sequence a) to d). 26.The peptide of claim 20, wherein the peptide sequence comprises asequence selected from the group consisting of: (SEQ ID NO: 24) VECTM XEKRVLA X ; (SEQ ID NO: 25) VECTX X EKRVLA X ; (SEQ ID NO: 26) VECTM XEKXVLA X ; (SEQ ID NO: 27) VECTX X EKXVLA X ; (SEQ ID NO: 28) VECTX XEKXVLA X LDKXXFLTQLHS; (SEQ ID NO: 29) VECTM X EKRVLA X LDKXXFLTQLHS;(SEQ ID NO: 30) VECTX X EKRVLA X LDKPPFLTQLHS; (SEQ ID NO: 31) VECTM XEKXVLA X LDKPPFLTQLHS;

wherein the residues which are bold and underlined X carry the staplingand is any amino acid derivative suitable for stapling; and wherein X isany amino acid except M, P and R.
 27. The peptide of claim 20, whereinthe peptide sequence comprises (SEQ ID NO: 32) VECTT R EKEVLA SLDKAAFLTQLHS;

wherein R and S carry the stapling; wherein the peptide optionallyfurther comprises from 1 to 5 or from 1 to 3 modification(s) of an aminoacid selected from substitution(s), deletion(s), addition(s), and amixture thereof.
 28. The peptide of claim 27, wherein R is2-(7-octenyl)arginine and S is 2-(4-pentenyl)serine.
 29. Apharmaceutical composition comprising a peptide according to claim 20.30. A method of treating a disease selected from the group consisting ofnonalcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver(NAFL), non-alcoholic steatohepatitis (NASH), hepatic steatosis (fattyliver), liver inflammation, cirrhosis, hepatocellular carcinoma andfibrosis comprising the administration of a peptide according to claim20, or a pharmaceutical composition thereof, to a subject in need oftreatment.
 31. The method of claim 30, wherein the fibrosis is a liverfibrosis, liver cirrhosis, renal fibrosis, cardiac fibrosis, atrialfibrosis, endomyocardial fibrosis, old myocardial infarction, pulmonaryfibrosis, cystic fibrosis, radio-induced lung fibrosis, vascularfibrosis, arterial fibrosis, brain fibrosis, myelofibrosis,arthrofibrosis, intestinal fibrosis, peritoneal fibrosis,retroperitoneal fibrosis or skin fibrosis.
 32. A pharmaceuticalcomposition comprising a peptide according to claim
 23. 33. A method oftreating a disease selected from the group consisting of nonalcoholicfatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL),non-alcoholic steatohepatitis (NASH), hepatic steatosis (fatty liver),liver inflammation, cirrhosis, hepatocellular carcinoma and fibrosiscomprising the administration of a peptide according to claim 23, or apharmaceutical composition thereof, to a subject in need of treatment.